sCD40L and placental growth factor (PLGF) as biochemical marker combinations in cardiovascular diseases

ABSTRACT

The invention relates to novel markers of vascular inflammation and combinations thereof as diagnostic and prognostic tools in patients with cardiovascular diseases. The markers also act as tools that facilitate the selection of active ingredients for the treatment of such diseases, and finally act as starting points for the treatment of cardiovascular diseases. Furthermore, the invention relates to the creation of an individual risk profile of negative events that are associated with the progression of arteriosclerosis.

This application is a continuation of application Ser. No. 10/534,985,which was filed May 16, 2005, and issued as U.S. Pat. No. 8,409,815which is a National Stage Entry under 35 USC §371 of InternationalApplication Number PCT/EP03/12531, filed on Nov. 10, 2003, which claimedpriority under 35 USC §119 to German Application No. 102 53 525.6, filedon Nov. 16, 2002, and to German Application No. 103 16 059.0, filed onApr. 8, 2003, all of which are incorporated by reference in theirentirety.

The invention relates to novel markers of vascular inflammation andcombinations thereof as diagnostic and prognostic tools in patients withcardiovascular diseases. The markers also act as tools that facilitatethe selection of active ingredients for the treatment of such diseases,and finally act as starting points for the treatment of cardiovasculardiseases. Furthermore, the invention relates to the creation of anindividual risk profile of negative events that are associated with theprogression of arteriosclerosis.

BACKGROUND OF THE INVENTION

The formation of a thrombus in the coronary vessel is the triggeringevent of an unstable coronary heart disease. In patients with anunstable coronary heart disease, the central role of the activation ofplatelets is further enhanced by thromboxane and prostaglandinmetabolites that are released from the platelets. Thus, the activationof platelets is a general therapeutic goal. Until now, such therapiescomprised the use of aspirin, tienopyridines and a direct glycoproteinIIb/IIIa-inhibitor. Nevertheless, until today, a reliable biochemicalmarker for the activation of platelets could not be identified. Resultswith P-selectin, the yet most promising marker for an activation ofplatelets, until today, are controversial.

Individuals that suffer from a cardiovascular disease can be groupedinto individuals that do not exhibit symptoms, and those that exhibitchest pain. The latter group can be grouped into individuals thatexhibit a stable angina pectoris (SAP), and those with acute coronarysyndromes (ACS). ACS patients can exhibit an unstable angina pectoris(UAP), or these patients already suffered from a myocardial infarction(MI). The MI can be an ST-elevated MI or a non ST-elevated MI. Theoccurrence of an MI can be associated by a left ventricular dysfunction(LVD). Finally, LVD patients experience a congestive heart failure (CHF)with a mortality rate of about 15%, or do not exhibit any symptoms.

Patients that are admitted with chest pain are analysed for an STincrease or depression. If this is the case, the individual will remainin hospital with a probability of nearly 100%. Since not all individualswith an MI exhibit ST-abnormalities, the troponin (TnT) level isdetermined, which in case of extraordinary high values indicates a highprobability that an MI has occurred.

Recent progresses in the basic research have established a fundamentalrole for the inflammation in the mediation of all phases of thearteriosclerosis, from its beginning through progression, and, finally,to the thrombotic complications of arteriosclerotic lesions [Libby P,Ridker P M, Maseri A. Inflammation and atherosclerosis. Circulation2002; 105(9): 1135-43. Ross R. Atherosclerosis—an inflammatory disease.N Engl J Med 1999; 340(2):115-26. Davies M J, Thomas A C. Plaquefissuring—the cause of acute myocardial infarction, sudden ischemicdeath, and crescendo angina. Br Heart J 1985; 53(4):363-73. Libby P.Molecular bases of the acute coronary syndromes. Circulation 1995;91(11):2844-50]. The results obtained from the association betweeninflammation and arteriosclerosis form the rational for the use ofcirculating inflammatory markers as potential predictive instruments inpatients with acute coronary syndromes. Indeed, elevated levels ofinflammatory markers, such as, for example, high sensitive C-reactiveprotein (hsCRP), serum amyloid A, and interleukin-6 (IL-6) are not onlyassociated with acute coronary syndromes in general [Berk B C, WeintraubW S, Alexander R W. Elevation of C-reactive protein in “active” coronaryartery disease. Am J Cordial 1990; 65(3):168-72, Biasucci L M, VitelliA, Liuzzo G, et al. Elevated levels of interleukin-6 in unstable angina.Circulation 1996; 94(5):874-7], but—what is more important—can alsopredict a statement regarding the clinical outcome of patients withacute coronary syndromes [Liuzzo G, Biasucci L M, Gallimore J R, et al.The prognostic value of C-reactive protein and serum amyloid a proteinin severe unstable angina. N Engl J Med 1994; 331(7):417-24, Biasucci LM, Liuzzo G, Grillo R L, et al. Elevated levels of C-reactive protein atdischarge in patients with unstable angina predict recurrentinstability. Circulation 1999; 99(7):855-60, Toss H, Lindahl B, SiegbahnA, Wallentin L. Prognostic influence of increased fibrinogen andC-reactive protein levels in unstable coronary artery disease. FRISCStudy Group. Fragmin during Instability in Coronary Artery Disease.Circulation 1997; 96(12):4204-10.]. Although the “classical” acute phaseprotein hsCRP is regarded as the most promising biomarker for clinicaluses, a substantial heterogeneity exists regarding the prevalence ofelevated hsCRP levels in patients with acute coronary syndromes[Biasucci L M, Liuzzo G, Colizzi C, Rizzello V. Clinical use ofC-reactive protein for the prognostic stratification of patients withischemic heart disease. Ital Heart J 2001; 2 (3): 164-71.].

Thus, more than 30% of the patients with severe unstable angina do notexhibit elevated hsCRP levels [Liuzzo G, Biasucci L M, Gallimore J R, etal. The prognostic value of C-reactive protein and serum amyloid aprotein in severe unstable angina. N Engl J Med 1994; 331(7): 417-24,Heeschen C, Hamm C W, Bruemmer J, Simeons M L. Predictive value ofC-reactive protein and troponin T in patients with unstable angina: acomparative analysis. CAPTURE Investigators. Chimeric c7E3 Anti-PlateletTherapy in unstable angina refractory to standard treatment trial. J AmColl Cardiol 2000; 35(6): 1535-42.]. In addition, individual differencesin the extent of the response to certain inflammatory stimuli possiblycan affect the levels of the “downstream” acute-phase reactants, such ashsCRP [Pepys M B, Hirschfield G M. C-reactive protein and its role inthe pathogenesis of myocardial infarction. Ital Heart J 2001, 2(11):804-6, Liuzzo G, Biasucci L M, Rebuzzi A G, et al. plasma proteinacute-phase response in unstable angina is not induced by ischemicinjury. Circulation 1996; 94(10): 2373-80.]. Therefore, still animportant challenge exists to identify proximal stimuli for vascularinflammation that can be used as risk-predicting serum markers inpatients with coronary arteriosclerosis.

In the meantime, there is increasing evidence that the CD40-CD40L-systemplays an important role in the pathophysiology of patients with unstablecoronary heart disease. Apart from the cell-associated form, CD40L alsooccurs in a soluble biologically completely active form, namely sCD40L.sCD40L is shedded by stimulated lymphocytes and actively released uponplatelet stimulation. sCD40L acts pro-inflammatory on endothelial cellsand promotes the coagulation, in that monocytes and endothelial cellsare stimulated to express tissue factors. In addition, sCD40L contains aKGD-sequence, a known binding motif that is specific for the predominantplatelet-integrin αIIbβ3. CD40L indeed is a αIIβ3-ligand, a plateletagonist, and is required for the stability of arterial thrombi. Anincrease of sCD40L can be detected in serum of patients with acutecoronary syndromes. It was reported (Schonbeck U, Varo N, Libby P,Buring J, Ridker P M. Circulation 2001; 104: 2266-8) that apparentlyhealthy women which exhibited elevated plasma concentrations of sCD40L,at the same time, carried an increased risk for cardiovascular events.

New findings show that a rupture of plaques and the subsequent formationof a thrombus in patients with acute coronary syndromes can lead to anactivation of the exposure of CD40L in circulating platelets (Lee Y, LeeW H, Lee S C, Ahn K J, Choi Y H, Park S W, Seo J D, Park J E.Cardiology. 1999; 92: 11-6). In addition, increased concentrations ofsCD40L were detected in patients with angina, whereby the concentrationswere particularly high in patients with unstable angina (Aukrust P,Muller F, Ueland T, Berget T, Aaser E, Brunsvig A, Solum N O, Forfang K,Froland S S, Gullestad L. Circulation 1999; 10: 614-20). These resultssuggest that a CD40L-CD40-interaction plays an important role during thepathogenesis of arteriosclerotic processes and the development ofcoronary syndromes.

Establishing the correct diagnosis associated with a suitable treatmentof patients with acute coronary syndromes that are not associated withan elevation of the ST-stretch can be very cumbersome. The exclusion ofacute myocardial infarction in accordance with actual standards isunsatisfactory. In the last years, a focussing on the riskstratification and control of the treatment has occurred with the aim toidentify patients wherein the risk exists to develop a life-threateningcardiologic event, and which, in particular, benefit from improvedtherapeutic and intervening strategies (Hamm C W, Bertrand M, BraunwaldE. Lancet 2001; 358:1533-8). In this respect, the ECG has only limitedprognostic relevance since important abnormities are rare and theirdetection is sparsely sensitive and specific (Kaul P, Fu Y, Chang W C,et al., J Am Coll Cardiol 2001; 38:64-71, and Savonitto S, Ardissino D,Granger C B, et al. JAMA 1999; 281: 707-13). Thus, markers of a necrosisof myocardial cells, in particular cardiac troponines, have developedinto valuable tools in the evaluation of patients with acute coronarysyndromes (Hamm C W, Braunwald E. Circulation 2000; 102: 118-22).Nevertheless, troponines are not actively involved in thepathophysiology of acute coronary syndromes, but rather represent a kindof surrogate markers for the fragile thrombus formation (Lindahl B,Diderholm E, Lagerqvist B, Venge P, Wallentin L. J Am Coll Cardio. 2001;38: 979-86, Heeschen C, van Den Brand M J, Hamm C W, Simoons M L.Circulation 1999; 100: 1509-14; Benamer H, Steg P G, Benessiano J, etal. Am Heart J 1999; 137: 815-20). In this respect, the ECG has onlylimited prognostic relevance since important abnormities are rare andtheir detection is sparsely sensitive and specific (Kaul P, Fu Y, ChangW C, et al., J Am Coll Cardiol 2001; 38:64-71 and Savonitto S, ArdissinoD, Granger C B, et al. JAMA 1999; 281:707-13).

Markers of an activation of platelets that determine the activity of thedisease, preferably before a myocardial necrosis occurs, could representimportant additional information for the diagnostic and therapeuticstratification in patients with acute coronary syndromes. There isincreasing evidence that also the CD40-ligand (CD40L, recently renamedinto CD154) plays an important role in the development of the diseaseand plaque-destabilisation (Mach F, Schonbeck U, Sukhova G K, AtkinsonE, Libby P. Nature 1998; 394: 200-3 and Lutgens E, Gorelik L, Daemen MJ, et al. Nat Med 1999; 5: 1313-6). The CD40-CD40L-system is common in amultitude of leukocytes and non-leukocytic cells, including endothelialcells and smooth muscle cells (Schonbeck U, Libby P. Cell Mol Life Sci2001; 58: 4-43), as well as in activated platelets (Henn V, Slupsky J R,Grafe M, et al. Nature 1998; 391: 591-4). In addition to thecell-associated 39-kDa form, CD40L also occurs in a soluble biologicallycompletely active form, namely sCD40L (Graf D, Muller S, Korthauer U,van Kooten C, Weise C, Kroczek R A. Eur J Immunol 1995; 25: 1749-54).sCD40L is shedded by stimulated lymphocytes and is actively releasedupon activation of platelets (Lee Y, Lee W H, Lee S C, et al. Cardiology1999; 92: 11-6, and Henn V, Steinbach S, Buchner K, Presek P, Kroczek RA. Blood 2001; 98: 1047-54). sCD40L acts pro-inflammatory on endothelialcells and promotes the coagulation by inducing the expression of tissuefactors by monocytes (Mach F, Schonbeck U, Bennefoy J Y, Pober J S,Libby P. Circulation 1997; 96: 396-9) and endothelial cells (Urbich C,Mallat Z, Tedgui A, Clauss M, Zeiher A M, Dimmeler S. J Clin Invest2001; 108: 1451-8). In addition, sCD40L contains a KGD-sequence (Graf D,Muller S, Korthauer U, van Kooten C, Weise C, Kroczek R A. Eur J Immunol1995; 25: 1749-54), a known binding motif that is specific for thepredominant platelet-integrin αIIbβ3 (Scarborough R M, Naughton M A,Teng W, et al. J Biol Chem 1993; 268: 1066-73). It could be shown thatCD40L indeed represents an αIIbβ3-ligand, a platelet agonist, and isrequired for the stability of arterial thrombi (Andre P, Prasad K S,Denis C V, et al. Nat Med 2002; 8: 247-52).

These findings stringently show that sCD40L plays an important role inthe pathophysiology of acute coronary syndromes. Interestingly, anincrease of sCD40L can be detected in serum of patients with acutecoronary syndromes (Aukrust P, Muller F, Ueland T, et al. Circulation1999; 100:614-20). It was reported that apparently healthy women thatexhibited elevated plasma concentrations of sCD40L carried an increasedrisk for cardiovascular events (Schonbeck U, Varo N, Libby P, Buring J,Ridker P M. Circulation 2001; 104: 2266-8). It is the aim of the presentinvention to examine the predictive value of sCD40L-concentrations withrespect to cardiac events and the curing effects of the glycoproteinIIb/IIIa-inhibitor abciximab in patients with acute coronary syndromes,wherein the data base of the CAPTURE-study (c7E3 Anti-Platelet Therapyin Unstable Refractory angina) was used (CAPTURE. Lancet 1997;349:1429-35).

Inflammatory markers that determine the activity of the disease,possibly before a myocardial necrosis occurs, can represent importantadditional information for the diagnostic and therapeutic stratificationin patients with acute coronary syndromes. The specific therapeuticinhibition of cytokines that are essential for the plaque-stability maybe a novel strategy for the treatment of patients with unstable andstable coronary heart disease.

It was recently shown for placental-growth factor (PlGF), a member ofthe family of the vascular-endothelial-growth factor-family(VEGF-family) of growth factors, that it is upregulated in early andprogressed arteriosclerotic lesions.

U.S. Pat. No. 6,225,088 describes PlGF in connection with proliferativediseases, whereas in the WO 92/06194, PlGF is described as anangiogenetic factor.

De Falco et al. (De Falco S, Gigante B, Persico M G. “Structure andfunction of placental growth factor” Trends Cardiovasc Med 2002 August;12(6):241-6) describe the association of derogated angiogenesis andarteriogenesis during pathologic conditions, such as, for example,ischemia and tumour formation in mice. Thereby, PlGF is described as anessential factor for the angiogenesis under pathologic conditions. PlGFis proposed as an alternative target for an angiogenetic therapy.

Luttun et al. (Luttun A, Tjwa M, Moons L, Wu Y, Angelillo-Scherrer A,Liao F, Nagy J A, Hooper A, Priller J, De Klerck B, Compemolle V, DaciE, Bohlen P, Dewerchin M, Herbert J M, Fava R, Matthys P, Carmeliet G,Collen D, Dvorak H F, Hicklin D J, Carmeliet P. Revascularization ofischemic tissues by PlGF treatment, and inhibition of tumourangiogenesis, arthritis and atherosclerosis by anti-Flt1 Nat Med 2002August; 8(8): 831-40) describe therapeutic methods of PlGF and Flt-1 inthe context of angiogenesis. It is furthermore described that inhibitionof PlGF reduces the growth and vulnerability of arterioscleroticplaques.

Oura et al. (Oura H, Bertoncini J, Velasco P, Brown L F, Carmeliet P,Detmar M. A critical role of placental growth factor in the induction ofinflammation and oedema formation. Blood 2003 Jan. 15; 101(2): 560-7)describe the role of PlGF in cutaneous inflammation and angiogenesis.Furthermore, the effects of elevated and reduced levels of PlGF andtheir comparison with the status of the inflammation are described.

The pro-inflammatory cytokine CD40L is released by activated platelets.The soluble form of CD40L, namely sCD40L, is increasingly present inpatients with acute coronary syndromes. Thus, the prognostic value ofsCD40L as a marker for the activation of platelets was also examined inview of the therapeutic effect of an inhibition of the glycoproteinIIb/IIIa-receptor.

Pregnancy-associated plasma protein-A (PAPP-A) is a high molecularweight zinc-binding matrix-metalloproteinase, belonging to themetzincin-superfamily of metalloproteinases, and was initiallyidentified in the plasma of pregnant women. It is broadly used for thescreening of foetal trisomy in the first trimester of gestation. It wasalso recently found for PAPP-A that it is expressed in eroded andloosened plaques, respectively, but is only minimally expressed instable plaques, and in those wherein a re-occurrence with symptoms as inpatients with ACS is assumed. Nevertheless, the exact role ofcirculating PAPP-A plasma levels for the prediction of hard endpoints,such as death or myocardial infarction, is not exactly determined inpatients with ACS. In addition, it is completely unknown whether PAPP-Aplasma levels provide additional prognostic information in patients withACS compared to recently established biomarkers. Therefore, theinventors compared the prognostic significance of PAPP-A plasma levelswith markers of systemic inflammation, the activation of platelets,ischemia, and myocardial necrosis in patients with ACS.

Many different molecular markers can be found in the state of the artthat can be suitable for the diagnosis of a cardiovascular disease.Examples of such markers are, amongst others:

Pregnancy-associated plasma protein A (PAPP-A); C-reactive protein(CRP); hs-CRP; placental growth factor (PlGF); interleukin-18(IL-18/IL-18b); brain natriuretic peptide (BNP); NT-pro brainnatriuretic peptide (NT-proNP); sCD40L, cTnI/T, IL-10, ICAM-1, VCAN-1,E-selectin, P-selectin, IL-6, VEGF, serum amyloid A (SAA), CKMB, MPO,LpPLAz, GP-BB, IL1RA, TAF1, soluble fibrin, anti-oxLDL, MCP-1, tissuefactor (TF), MMP-9, Ang-2, Tffi-2, IL-P, bFGF, PCM, and VEGF-A.

Some of the above indicated markers are markers known and characterisedfor the examination of coronary diseases, nevertheless, others have notyet been correspondingly examined.

Nearly all of the given markers have a diagnostic value with respect tocertain cardiovascular events. TnT, for example, is of particular valuefor the diagnosis and the prediction of MI (see above). Inflammatorymarkers, such as CRP, are valuable for the diagnosis and prediction ofan inflammation that can lead to a plaque-rupture and MI.

Many of the above indicated markers have a diagnostic value forcardiovascular diseases. The use of a combination of markers wasdescribed only very conservatively.

Lund et al. in Circulation. 2003, 108:1924-1926, describe thecombination of PAPP-A with TnI (troponin I); Peng et al. in ClinicaChimica Acta 319 (2002) 19-26, describe the combination of sCD40L withsICAM-1 and sVCAM-1. Lenderink et al. in European Heart Journal (2003)24, 77-85, describe the combination of TnT with CRP. Heeschen et al. inJournal of the American College of Cardiology; Vol. 35, No. 6, 2000,mention the combination of TnT with CRP.

Heeschen et al. in Circulation. 2003; 107:2109-2114, describe thecombination of TnT, CRP and IL-10. Blankenberg et al. in Circulation.2002; 106:24-30, describe the combination of CRP and IL-6. Autiero etal. in Journal of Thrombosis and Haemostasis, 1:1356-1370, describe thecombination of PlGF and VEGF.

However, until now the potential of the improved analysis by thecombination has neither been examined, nor was the development ofselective superior marker-assays completed. Which markers are suitablefor an effective diagnosis therefore can not be readily derived from theabove indicated publications.

In view of the above, it is therefore an object of the present inventionto provide a method by which the risk of an adverse cardiovascular eventbeing due to coronary thrombosis can be estimated with the aid of anindividual risk profile. It is a further object of the present inventionto develop a method for the evaluation of the probability, whether atreatment with an active ingredient for the inhibition of the placentalgrowth factor (PlGF) is advantageous. With the aid of this method theattending physician shall be, better than before, enabled to selectsuitable measures in order to positively influence the patients and/orto prevent an adverse event, or to at least reduce it in its severityfor the affected patients.

It is the object of the present invention to develop a method by whichthe risk to suffer from an adverse cardiovascular event being due to acoronary thrombosis can be estimated with the aid of an individual riskprofile. This shall be done by measuring the concentration of a markerof the activation of platelets. It is furthermore an object of thepresent invention to develop a method for evaluating the probability,whether a treatment with an active ingredient for the inhibition of theactivation of platelets is advantageous. With the aid of this method theattending physician shall be, better than before, enabled to selectsuitable measures in order to positively influence the patients and/orto prevent an adverse event, or to at least reduce it in its severityfor the affected patients.

It is a further object of the present invention to find generalcombinations of diagnostic markers for cardiovascular events that allowfor a precise diagnosis which cardiovascular event the patient hadalready suffered from, and from which he will possibly suffer in thefuture. It is a particular object to find marker combinations that canadvantageously be performed in parallel. Ideally, these measurementswould allow for measuring patients with chest pain, simultaneously withTnT.

The object of the present invention is solved by a method for analysingsamples in association with acute cardiovascular diseases. The methodaccording to the invention comprises the steps of: (a) obtaining abiological sample to be analysed from a subject; (b) determining of theconcentration of at least one marker selected from soluble CD40-ligand(sCD40L), PAPP-A, and PlGF, (c) optionally, determining of theconcentration of at least one additional marker selected from troponin T(TnT), MPO, NT-proBNP, VEGF, BNP, and additional inflammatory markers,and (d) comparing the results that are obtained for the sample to beanalysed with reference value/s and/or the values from referencesamples.

The markers that are used for the analysis in the context of the presentinvention are, to the largest part, markers that are well known andcharacterised from the state of the art, that, however, until now wereonly insufficiently characterised for the use in a diagnosis of an acutecardiovascular disease.

Thus, C-reactive protein (CRP) and hsCRP are characterised as markers ofsystemic inflammation, troponin cTn1/T is characterised as marker fornecrosis; the pregnancy associated plasma protein A (PAPP-A) ischaracterised as a marker for the activation of macrophages; IL-10(Interleukin 10) is characterised as a marker for the inflammatorybalance, sCD40L is characterised as a marker for thethrombo-inflammatory activation, MPO (myeloperoxidase) is characterisedas a marker for oxidative stress, placental growth factor (PlGF) ischaracterised as a marker for vascular inflammation, and the markersbrain natriuretic peptide (BNP) and NT-pro brain natriuretic peptide(NT-proNP) are characterised as markers of neurohumoral activation andischemia.

Other similar and also useful markers are interleukin-18 (IL-18/IL-18b),ICAM-1, VCAN-1, E-selectin, P-selectin, IL-6, VEGF, serum amyloid A(SAA), CKMB, LpPLAz, GP-BB, IL-1RA, TAF-1, soluble fibrin, anti-oxLDL,MCP-1, tissue factor (TF), MMP-9, Ang-2, Tffi-2, bFGF, PCM, and VEGF-A.

WO 03/040692 (and Circulation 2001; 104:2266-2268) describes that sCD40Lcan be advantageously used in combination with an inflammatory marker,in particular CRP. However, this combination is exclusively used for thediagnosis of non-acute cardiac diseases, due to doubtful results astratification is rejected by WO 03/040692.

Preferred is a method according to the invention, wherein the sample tobe analysed and/or the reference sample is derived from a mammal, inparticular from a human. Further preferred is a method according to theinvention, wherein the sample to be analysed and/or the reference sampleis selected from the group consisting of peripheral blood or fractionsthereof, and cell culture suspensions or fractions thereof. It iffurther preferred that the sample to be analysed and/or the referencesample is blood serum or blood plasma. Peripheral whole blood isparticularly preferred as a sample to be analysed and/or referencesample.

According to a further aspect of the method according to the invention,the sample to be analysed and/or the reference sample can bepre-treated, wherein, e.g., a coagulation inhibitor, in particularheparin, is added to said peripheral blood.

In accordance with the invention, it could surprisingly be found that,in addition to the individual analysis of several of the above-mentionedmarkers, also combinations of such markers are possible for a diagnosisor monitoring of acute cardiac events, allowing for a markedly improvedanalysis. In doing so, combinations of markers could be identified asparticularly preferred that relate to different aspects of the adversecardiac event, but can be analysed simultaneously (that is,simultaneously or in a more or less timely spaced series ofmeasurements). In one aspect of this invention, additional markers areselected from inflammatory markers, such as, for example, from CRP,(hs)CRP, and IL-10.

According to a further aspect of the method according to the invention,the markers and combinations thereof as analysed are selected fromsCD40L; PAPP-A; PlGF; sCD40L+TnT; PAPP-A+TnT; PlGF+TnT; sCD40L+PAPP-A;sCD40L+PlGF; PAPP-A+PlGF; sCD40L+PAPP-A+TnT; sCD40L+PlGF+TnT;PAPP-A+PlGF+TnT; sCD40L+PAPP-A+PlGF; and sCD40L+PAPP-A+PlGF+TnT. Then,preferred is the further combination with at least one of the additionalmarkers MPO, NT-proBNP, BNP, CRP, (hs)CRP, and IL-10.

Then, particularly preferred according to the invention is a method,wherein the markers and combinations thereof as analysed are selectedfrom CRP, TnT, PAPP-A; CRP, TnT, PAPP-A, IL-10; CRP, TnT, PAPP-A, IL-10,sCD40L, and TnT, PAPP-A, IL-10, sCD40L, VEGF.

The method as used according to the invention for determining theconcentrations of the markers as analysed can be selected from allsuitable methods for detecting proteins in biological samples that areknown to the person of skill in the art. In the context of the presentinvention, the actual method is not important as long as the method issensitive enough in order to fall below the detection level that isrequired for an accurate determination of the concentrations of themarkers. Suitable methods are normally based on the binding of a labelto the marker to be determined, and the subsequent detection of thislabel. Thereby, the binding can be covalent or non-covalent and/or occurdirectly or indirectly. Suitable methods for measuring according to thepresent invention include, e.g., electro-chemiluminescence.Turbidimetry, nephelometry, and latex-enhanced turbidimetry ornephelometry can also be used.

Due to its high sensitivity and the fact that these methods can also beadapted to highthroughput-environments, according to the inventionmethods are preferred, wherein the determining of the concentrationtakes place by means of an immunological method by means ofmarker-binding molecules. Examples for such methods are ELISA(enzyme-linked immunosorbent assay), sandwich enzyme immunoassays orsolid-phase immunoassays. Preferred is therefore that the marker-bindingmolecules are selected from the group consisting ofantimarker-antibodies or parts thereof, and marker-receptors or partsthereof.

These molecules can be selected from a very large multitude ofmarker-specific molecules. It is preferred that the marker-bindingmolecules are selected from the group consisting of antibodies that arespecifically directed against markers or against parts thereof, or partsor fragments thereof, and a marker-receptor or parts thereof, or anintegrin, e.g. the platelet-integrin αIIbβ3 or parts thereof.Particularly preferred is a method according to the invention, whereinthe antibodies, parts or fragments thereof comprise polyclonalantibodies, monoclonal antibodies, Fab-fragments, scFv-antibodies, anddiabodies.

According to a further aspect of the method of the present inventioncomponents of the method can be present bound to a solid phase, thus,the marker-binding molecules can be present in solution ormatrix-immobilised. A multitude of materials that are known to theperson of skill are used as matrices, such as, for example,resin-matrices and/or common columnmatrices. Particularly preferred isfurthermore a method according to the invention, wherein themarker-binding molecules are coupled to one or several detectionmolecules from the group consisting of fluorescein thioisocyanate,phycoerythrine, enzymes (for example horseradish-peroxidase), andmagnetic bead.

According to a further aspect of the method according to the invention,the marker-binding molecules can be detected with an antibody that iscoupled to one or several detection molecules. Thus, this represents anindirect detection of the binding of the molecule. Such two-stepdetections are very well known to the person of skill, for example, fromthe technology of anti-antibody-detection.

According to a further aspect of the method of the present invention,immunocytological methods can be used for the analysis of the sample.For this, all methods are suitable that allow for a specificdetermination based on the marker/molecule-interaction. Preferred aremethods that are selected from the group consisting ofsandwich-enzyme-immunoassay, ELISA, and solid phase immunoassays.

The results that are obtained for the samples to be analysed are usuallycompared with a reference sample. Which sample can serve as a referencesample will, in particular, depend from the kind of the sample that isanalysed, and the history of the disease of the individual from whichthe sample to be analysed is derived. Preferred is a method according tothe invention, wherein the reference sample is derived from one or themean value of several mammals, wherein a cardiovascular disease wasexcluded. Nevertheless, this is not mandatory, if, e.g. the progressionof a disease shall be determined, also an “old” sample of the samepatient can be used as a reference sample. It will be obvious for theperson of skill, which samples are suitable as reference samples for themethod according to the invention.

According to a further aspect of the method according to the presentinvention, the acute cardiovascular diseases that are to be diagnosedand/or prognosed, and/or whose therapy shall be monitored can beselected from the group consisting of unstable angina, myocardialinfarction, acute heart syndromes, coronary arterial disease, and heartinsufficiency. Nevertheless, it shall not be excluded that the methodaccording to the invention is suitable for and can be employed inadditional acute cardiac disease conditions.

A further aspect of the present invention relates to a diagnostic kit,wherein said kit comprises means for performing the method according tothe invention, optionally together with additional components and/orexcipients. Preferably, such means are at least one antibody fordetecting of markers, and means for the subsequent quantification ofsaid markers. In addition, the kit can contain other components and/orenzymes for performing the method according to the present invention,e.g. instruction manuals for an interpretation of the results of theassays in view of the risk profile of the patient, and correspondingcountermeasures and proposals for therapy.

It is preferred to perform the method according to the invention withthe aid of a diagnostic kit that comprises gold-labelled polyclonalmouse-indicator antibodies, biotinylated polyclonal detectionantibodies, and an assay device comprising a fibreglass-fleece.

A further aspect of the present invention thus relates to the use of themethod according to the invention for the diagnosis and/or prognosis ofacute cardiovascular diseases, and/or for monitoring of their therapy.This is done by the quantitative and critical determination of markers.Based on the risk profile that then can be generated, suitablecountermeasures can then be performed by the attending physician inorder to positively influence the patients and to prevent the adverseevent or at least to reduce it in its severity for the affected patient.Such a therapy according to the invention can, e.g., comprise theadministration of statines or inhibitors of the glycoproteinIIb/III-receptor, in particular abciximab. However, the person of skillis aware of further possible therapies in accordance with a commonscheme in order to treat cardiovascular diseases that can occur.

In a further embodiment of the invention, an anti-inflammatory means isco-administered. Said means can be selected from non-steroid or steroidanti-inflammatory means that, e.g., can include: alclofenac;alclometason; dipropionate; algestonacetonide; alpha-amylase; amcinafal;amcinafid; amfenac sodium; amiprilose hydrochloride; anakinra; anirolac;anitrazafen; apazon; balsalazid disodium; bendazac; benoxaprofen;benzydamine hydrochloride; bromelain; broperamol; budesonide; carprofen;cicloprofen; cintazon; cliprofen; clobetasolpropionate;clobetasonbutyrate; clopirac; cloticasonpropionate; cormethasonacetate;cortodoxon; deflazacort; desonid; desoximetason;dexamethasondipropionate; diclofenac potassium; diclofenac sodium;diflorasondiacetate; diflunudon sodium; diflunisal; difluprednat;diftalon; dimethylsulfoxide; drocinonid; endryson; enlimomab; enolicamsodium; epirizol; etodolac; etofenamat; felbinac; fenamol; fenbufen;fenclofenac; fenclorac; fendosal; fenpipalon; fentiazac; flazalon;fluazacort; flufenamine acid; flumizol; runisolidacetate; plunixin;flunixin meglumine; fluocortin butyl; fluorometholonacetate; fluquazon;flurbiprofen; fluretofen; fluticasonpropionate; puraprofen; furobufen;halcinonid; halobetasolpropionate; halopredonacetate; ibufenac;ibuprofen; ibuprofen aluminium; ibuprofen piconol; ilonidap;indomethacin; indomethacin sodium; indoprofen; indoxol; mitrazol;isoflupredonacetate; isoxepac; isoxicam; ketoprofen; lofemizolhydrochloride; lomoxicam; loteprednol etabonat; meclofenamat sodium;meclofenamine acid; meclorison dibutyrate; mefenamin acid; mesalamine;meseclazon; methylprednisolon suleptanate; momiflumat; nabumeton;naproxen; naproxen sodium; naproxol; nimazon; olsalazin sodium;orgotein; orpanoxin; oxaprozin; oxyphenbutazon; paranylin hydrochloride;pentosan polysulfat sodium; phenbutazon sodium glycerate; pirfenidon;piroxicam; piroxicam cinnamate; piroxicam olamine; pirprofen; prednazat;prifelon; prodolinic acid; proquazon; proxazol; proxazolcitrate;rimexolon; romazarit; salcolex; salnacedin; salsalat; salicylates;sanguinariumchloride; seclazon; sermetacin; sudoxicam; sulindac;suprofen; talmetacin; talniflumat; talosalat; tebufelon; tenidap;tenidap sodium; tenoxicam; tesicam; tesimid; tetrydamine; tiopinac;tixocortol pivalat; tolmetin; tolmetin sodium; triclonid; triflumidate;zidometacin; glucocorticoides; zomepirac sodium.

In the context of the present invention, “diagnosis” relates to theascertaining, whether an individual has suffered from a particularcardiovascular event. In the context of the present invention,“prognosis” relates to the prediction of the probability (in %) whetheran individual will suffer from a particular cardiovascular event. In thecontext of the present invention, “stratification of the therapy”relates to the determination of the suitable therapeutic treatment forsaid cardiovascular event that will occur or has occurred. In thecontext of the present invention, “monitoring of the therapy” relates tothe control and, optionally, an adjusting of the therapeutic treatmentfor an individual. In the context of the present invention, “therapeutictreatment” includes all treatments that possibly improve thepathophysiological condition of an individual, and, e.g., includes theadministration of pharmaceutics as well as chirurgical treatment (e.g.balloon dilatation).

Base line-values of the sCD40L-concentration make available informationof prognostic value in patients with acute coronary syndromes,independently of the occurrence of a myocardial necrosis. In addition,the high risk group of patients that can gain the largest benefit froman anti-platelet-treatment with abciximab can be identified by means ofsCD40L.

The present study provides direct evidence for the fact that sCD40L isan informative biochemical marker of an activation of platelets. Anincrease of the concentration of CD40L reliably identifies a specificsubgroup of patients with acute coronary syndromes that carry a profoundrisk to suffer from a cardiac event, and that gain the largest benefitfrom treatment with the glycoprotein IIb/IIIa-receptor-antagonistabciximab. Correspondingly, CD40L does not only essentially contributeto the pathophysiology of acute coronary syndromes, but provides areliable and informative clinical marker by which patients having aformation of high risk-lesions and/or coronary thrombosis can beidentified (Andre P, Prasad K S, Denis C V, et al., Nat Med 2002;8:247-52; Andre P., Nannizzi-Alaimo L, Prasad S K, Phillips D R.;Circulation 2002; 106: 896-9).

In 40.5% of the CAPTURE-patients, the sCD40L-concentrations in thecirculation were present above the calculated threshold concentration of5.0 μg/l. These patients having an elevated sCD40L-concentrations carrya profound cardiac risk to experience a lethal or non-lethal myocardialinfarction. This elevated cardiac risk in patients with highsCD40L-concentrations receiving a placebo was particularly obviousduring the first 72 hours (FIG. 5 a). However, the frequency of theevents during the whole six months developed further apart (FIG. 5 b).sCD40L could be found as an informative prognostic marker beingindependent from the detection of a myocardial necrosis and independentfrom the inflammatory markers CRP and TNF-α as well as from the adhesionmolecule ICAM-1. TnT, CRP, and sCD40L provided reliable prognosticinformation in a multivariate regression model (Table 2). Using thesCD40L-concentration, patients without indications of a myocardialnecrosis were identified that exhibited an increased cardiac risk. Byusing predetermined threshold concentrations for TnT and CRP togetherwith a classification of the patients based on the number of cardiacmarkers that were elevated it was found that a simultaneous estimationof these pathobiologically different biochemical markers at the timewhere the patient is admitted allows for an informative prediction ofthe risk of the patient to suffer from an adverse cardiac event duringthe following six months.

Troponines represent markers of a myocardial necrosis, nevertheless,they are not actively involved in the pathophysiology of acute coronarysyndromes. Rather, they are surrogate markers for the fragile formationof thrombi (Lindahl B, Diderholm E, Lagerqvist B, Venge P, Wallentin L;J Am Coll Cardiol 2001; 38:979-86; Heeschen C, van Den Brand M J, Hamm CW, Simoons M L; Circulation 1999; 100:1509-14; Benamer H, Steg P G,Benessiano J, et al.; Am Heart J 1999; 137:815-20). In postmortem-studies on patients with acute coronary syndromes, an erosion orrupture of the fibrous caps of the arteriosclerotic plaques that are thebasis of the pathophysiology was identified (Lindahl B, Diderholm E,Lagerqvist B, Venge P, Wallentin L; J Am Coll Cardiol 2001; 38:979-86;Heeschen C, van Den Brand M J, Hamm C W, Simoons M L; Circulation 1999;100:1509-14). An exposure of components of the plaques, collagen andother components of the vascular wall leads to an increase of thevascular tonus and the activation of platelets (Farb A, Burke A P, TangA L, et al.; Circulation 1996; 93:1354-63; Davies K T, Thomas A C; BrHeart J 1985; 53:363-73; Davies M J; N. Engl J Med 1997; 336:1312-4).The thrombotic embolism of a coronary artery with increasedmicrovascular perfusion and necrosis is an essential component of acutecoronary syndromes (Heeschen C, van Den Brand M J, Hamm C W, Simoons ML; Circulation 1999; 100:1509-14; Benamer H, Steg P G, Benessiano J, etal.; Am Heart J 1999; 137:815-20). Correspondingly, sensitive markers,in particular troponines as surrogate markers of an arterial thromboticembolism that results from an active thrombotic process in theunderlying lesion, serve for the detection of a small injury of themyocardium.

In contrast to this, sCD40L could be directly involved in thepathophysiology of acute coronary syndromes in several ways. Recentevidences suggest that CD40L contributes essentially to the progressionof an arteriosclerosis, and correspondingly to a destabilisation ofarteriosclerotic plaques (Mach F, Schonbeck U, Sukhova G K, Atkinson E,Libby P; Nature 1998; 394:200-3; Lutgens E, Gorelik L, Daemen M J, etal.; Nat Med 1999; 5:1313-6). It was proposed thatCD40/CD40L-interactions promote complications by atheroms in that theyinduce the expression of cytokines, chemokines, growth factors,matrix-metalloproteinases, and procoagulants in differentatherom-associated cellular types (Schonbeck U, Libby P.; Cell Mol LifeSci 2001; 58:4-43; Henn V, Slupsky J R, Grafe M, et al.; Nature 1998;391:591-4; Henn V, Steinbach S, Buchner K, Presek P, Kroczek R A; Blood2001; 98:1047-54; Mach F, Schonbeck U, Bonnefoy J Y, Pober J S, LibbyP.; Circulation 1997; 96:396-9; Miller D L; Yaron R, Yellin M J; JLeukoc Biol 1998; 63-373-9; Kotowicz K, Dixon G L, Klein N J, Peters MJ, Callard R E; Immunology 2000; 100:441-8). Novel studies have shownthat, in addition to leukocytes and non-leukocytic cells includinggranulocytes, mononuclear phagocytes, endothelial cells and cells of thesmooth musculature (Schonbeck U, Libby P; Cell Mol Life Sci 2001;58:4-43), activated platelets produce and release large amounts ofsCD40L (Henn V, Steinbach S, Buchner K, Presek P, Kroczek R A; Blood2001; 98:1047-54). Another study shows that a cardiopulmonary bypasscauses an increase of the concentration of sCD40L in plasma thatcorresponds to a decrease of the content of CD40L in platelets,suggesting that sCD40L primarily is derived from the platelets and couldcontribute to the thrombotic complications that are related to such abypass (Nannizzi-Alaimo L, Rubenstein M H, Alves V L, Leong G Y,Phillips D R, Gold H K; Circulation 2002, 105:2849-2854). It wasfurthermore found that sCD40L positively correlates with solubleP-selectin in plasma and 11-dehydrothromboxane B₂-concentrations inurine (Cipollone F, Mezzetti A, Porreca E, et al.; Circulation 2002;106:399-402). In addition, experimental analyses showed that CD40L isrequired for a stabilisation of arterial thrombi (Andre P, Prasad K S,Denis C V et al.; Nat Med 2002; 8:247-52). The present study nowprovides direct evidence for the fact that CD40L indeed is a marker ofthe activation of platelets. The activation of platelets as determinedby flow cytometry in patients with acute coronary syndromes correlatedsignificantly with the concentrations of sCD40L in serum (FIG. 7).Interestingly, sCD40L was found as an independent predictive factor ofan activation of platelets with the highest significance. The results ofthe present study established sCD40L-concentrations as highlyinformative prognostic markers in patients with acute coronary syndromesthat are in danger to suffer from a thrombosis. These findings aresupported by the fact that an inhibition of the glycoproteinIIb/IIIa-receptor by abciximab eliminated the increased risk in patientswith acute coronary syndromes and elevated sCD40L-concentrations. Whiletroponin positively indicated the tendency of a thrombus to cause anembolism and to lead to myocardial necrosis, elevated concentrations ofsCD40L in patients with acute coronary syndromes reflect the thromboticactivity of the triggering lesion to recruit and activate platelets.

It was shown in an earlier analysis of a subgroup of patients of theCAPTURE-study that an additional treatment with the glycoproteinIIb/IIIa-receptor-antagonist abciximab reduced the elevated risk oftroponin-positive patients to the extent of troponin-negative patients(Hamm C W, Heeschen C, Goldmann B, et al.; N Engl J Med 1999;340:1623-9). These patients constitute about ⅓ of the patients withacute coronary syndromes (Hamm C W, Braunwald E; Circulation 2000;102:118-22; Antman E M, Tanasijevic M J, Thompson B, et al; N Engl J Med1996; 335:1342-9; Ohman E M, Armstrong P W, Christenson R H, et al.; NEngl J Med 1996; 335:1333-41; Hamm C W, Ravkilde J, Gerhardt W, et al.;N Engl J Med 1992; 327:146-50; Hamm C W, Goldmann B U, Heeschen C,Kreymann G, Berger J, Meinertz T; N Engl J Med 1997; 337:1648-53).Similar findings regarding troponin T and troponin I later resulted fromother studies (Newby L K, Ohman E M, Christenson R H, et al.;Circulation 2001; 103:2891-6; Januzzi J L, Chae C U, Sabatine M S, JongI K; J Thromb Thrombolysis 2001; 11:211-5; Heeschen C, Hamm C W,Goldmann B, Deu A, Langenbrink L, White H D; Lancet 1999; 354:1757-62),and troponines were subsequently included into the novel guidelines aspart of the risk stratification in patients with acute coronarysyndromes (Hamm C W, Bertrand M, Braundwald E; Lancet 2001; 358:1533-8;Braunwald E, Maseri A, Armstrong P W, et al.; Eur Heart J 1998;19:D22-30). It is shown in the present study that such a pronouncedpositive effect of an anti-platelet-therapy is obvious also in patientswith elevated sCD40L-concentrations. The present analysis suggests thatpatients with acute coronary syndromes that exhibit increasedconcentrations of sCD40L are effectively stabilised by the glycoproteinIIb/IIIa-receptor-antagonist abciximab (FIGS. 5 a, b). At a calculatedthreshold concentration of 5.0 μg/l, an abrupt change of the riskquotient of 0.87 for the second quintile, and 1.12 for the thirdquintile towards significantly lower values of 0.36 for the fourthquintile, and 0.38 for the fifth quintile, respectively, (FIG. 4) wasobserved. Interestingly, the concentrations of TnT and sCD40L providedindependent predictive values with respect to both the risk of ischemicevents as well as the positive effect of a glycoproteinIIb/IIIa-receptor-inhibition by abciximab. Patients without indicationsof a myocardial injury (troponin-increase is lacking) which,nevertheless, exhibited increased concentrations of sCD40L, gained asubstantial benefit from the treatment with the glycoproteinIIb/IIIa-inhibitor abciximab. Thus, patients with a high risk for athrombosis of the coronary vessels, as proven either by an increase ofthe sCD40L-concentration or an increase of the TnT-concentration, whichfinally made up 54% of the overall patients involved the CAPTURE-study,had a pronounced advantage from the treatment with abciximab, with arisk quotient of 0.38 [0.21-0.72]; p<0.001).

In summary, it can be stated that the present study documents theimportant and independent role of sCD40L as a marker of the activationof platelets for the diagnostic and therapeutic risk stratification. Theincreased cardiac risk of patients with high sCD40L-concentrations thatreceived a standard therapy with heparin and aspirin was reverted by theglycoprotein IIb/IIIa-receptor-antagonist abciximab. The combined use oftroponines and sCD40L that both represent essential components of thepathophysiology in patients with acute coronary syndromes providesimportant insights into the activity of the disease, the cardiologicrisk, and the effectiveness of a treatment by means of glycoproteinIIb/IIIa-inhibition by abciximab being superior to the use of a singlemarker.

The results of the present study establish the PlGF serum level as anovel and effective independent prognostic determinant of the clinicaloutcome in patients with acute coronary syndromes. It has to beparticularly noted that in patients with lower hsCRP serum levelselevated PlGF serum levels identify a subgroup of patients that sufferfrom a significantly increased cardiac risk (fitted hazards-ratio 3.58[95% CI 1.48-7.72]; p=0.001).

The predictive value of PlGF serum levels is independent from evidencefor myocardial necrosis as determined by the troponin serum level.Finally, elevated PlGF serum levels not only identify those patientswith acute chest pain that develop acute coronary syndromes, but alsothose patients that suffer from an increased risk of reoccurringinstability from an initial acute coronary syndrome after discharge.Thus, measuring of the PlGF serum levels can not only be a reliable andeffective clinical tool for the identification of patients with highrisk formation of lesions but also of persistent vascular inflammationof the coronary circulation.

The role of PlGF as a primary inflammatory marker of the instability ofarteriosclerotic lesions can be explained by its well documentedpro-inflammatory effects in animal models of arteriosclerosis orarthritis [Luttun A, Tjwa M, Moons L, et al. Revascularization ofischemic tissues by PlGF treatment, and inhibition of tumorangiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat Med 2002;8(8):831-40]. Although PlGF belongs to the family of VEGF, itsetiopathogenetic role appears to be associated rather with inflammationas with angiogenesis [Luttun A, Tjwa M, Moons L, et al.Revascularization of ischemic tissues by PlGF treatment, and inhibitionof tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. NatMed 2002; 8(8):831-40.]. Indeed, whilst the VEGF-increase due to hypoxiaand the increase of the VEGF-serum level are regarded as an earlyadaptation of the myocardium to the decreasing bloodflow [Lee S H, WolfP L, Escudero R, Deutsch R, Jamieson S W, Thistlethwaite P A. Earlyexpression of angiogenesis factors in acute myocardial ischemia andinfarction. N Engl J Med 2000; 342(9):626-33.], PlGF is not affected ordown-regulated by hypoxia [Khaliq A, Dunk C, Jiang J, et al. Hypoxiadown-regulates placenta growth factor, whereas fetal growth restrictionup-regulates placenta growth factor expression: molecular evidence for“placental hyperoxia” in intrauterme growth restriction. Lab Invest1999; 79(2):151-70, Cao Y, Linden P, Shima D, Browne F, Folkman J. Invivo angiogenic activity and hypoxia induction of heterodimers ofplacenta growth factor/vascular endothelial growth factor. J Clin Invest1996; 98(11):2507-11.]. In agreement with these data, the results ofthis study do not result in a correlation between the PlGF serum leveland the troponin T serum level as a marker of myocardial necrosis,whereas the VEGF serum level positively correlated with the troponin Tserum level. In agreement with this, PlGF did not correlate with theVEGF serum level.

Therefore, the PlGF serum level appears not to be affected by myocardialnecrosis. In contrast, the VEGF serum levels are coupled with anincrease of troponin T, affected TIMI flow, and clinical signs ofmyocardial ischemia [Heeschen C, Dimmeler S, Hamm C W, Boersma E, ZeiherA M, Simoons M L. Prognostic significance of angiogenic growth factorserum levels in patients with acute coronary syndromes. Circulation2003; 107:524-530.]. The PlGF serum level being insensitive versussmaller myocardial injuries could specifically be important in patientswith acute coronary syndromes, out of which about one third atpresentation are positive for troponin [Hamm C W, Braunwald E. Aclassification of unstable angina revisited. Circulation 2000; 102(1):118-22.].

Similarly, a myocardial injury could also compromise the value of thehsCRP serum level in order to be able to predict the outcome in patientswith acute coronary syndromes. As a classical unspecific downstreamacute-phase-marker, the hsCRP serum levels in patients with myocardialinjury are increased as measured by an increase of troponin T. It iswell established that elevated serum levels of hsCRP are found beforethe occurrence of a marker of myocardial necrosis in nearly all patientsin which an unstable angina occurs before an infarction [Liuzzo G,Baisucci L M, Gallimore J R, et al. Enhanced inflammatory response inpatients with preinfarction unstable angina. J Am Coll Cardiol 1999;34(6): 1696-703.]. The specificity of elevated hsCRP serum levelconfirming an enhanced vascular inflammation in the presence ofmyocardial injury is therefore very limited.

Thus, elevated hsCRP serum levels in troponin-positive patients can onlyrepresent an elevated risk secondary to myocardial injury as a surrogatemarker for thrombotic embolism that rather is derived from an activethrombotic process within the culpritic lesion than from a persistingvascular inflammation.

Indeed, when each of troponin T and VEGF as markers of myocardialnecrosis and ischemia are included into a multivariate analysis,elevated hsCRP serum levels are no longer predictive for an elevatedrisk in patients with acute coronary syndromes.

More important, the reported prevalence of elevated hsCRP serum levelsvaries considerably in acute coronary syndromes. (more than 30% of thepatients with severe unstable angina and more than 50% of the patientswith an acute myocardial infarction do not exhibit elevated hsCRP serumlevels). Elevated hsCRP serum levels are lacking in more than 30% ofpatients with severe unstable angina and in more than 50% of those withan acute myocardial infarction that does not follow after an unstableangina [Liuzzo G, Baisucci L M, Gallimore J R, et al. Enhancedinflammatory response in patients with preinfarction unstable angina. JAm Coll Cardiol 1999; 34(6): 1696-703.] suggesting an importantheterogenicity of the role of inflammatory triggers of the clinicalsyndrome of the coronary instability [Libby P, Ridker P M, Maseri A.Inflammation and atherosclerosis. Circulation 2002; 105(9): 1135-43.].However, it is also well established that individuals can vary in theirsystemic responses towards a particular inflammatory stimulus [Liuzzo G,Buffon A, Biasucci L M, et al. Enhanced inflammatory response tocoronary angioplasty in patients with severe unstable angina.Circulation 1998; 98(22):2370-6, Liuzzo G, Angiolillo D J, Buffon A, etal. Enhanced response of blood monocytes to in vitrolipopolysaccharide-challenge in patients with recurrent unstable angina.Circulation 2001; 103(18):2236-41. Biasucci L M, Vitelli A, Liuzzo G, etal. Elevated levels of interleukin-6 in unstable angina. Circulation1996; 94(5):874-7.]. The increase in hsCRP or IL-6 that was observed inresponse to the vascular trauma induced by balloon dilatation or even byuncomplicated cardiac catheterisation correlates linearly with baselinehsCRP or interleukin-6 serum levels [Liuzzo G, Buffon A, Biasucci L M,et al. Enhanced inflammatory response to coronary angioplasty inpatients with severe unstable angina. Circulation 1998; 98(22):2370-6].In addition, the IL-6 production by monocytes that are isolated frompatients with unstable angina significantly increased in patients withelevated hsCRP serum levels, compared to patients with normal hsCRPserum levels [Liuzzo G, Angiolillo D J, Buffon A, et al. Enhancedresponse of blood monocytes to in vitro lipopolysaccharide-challenge inpatients with recurrent unstable angina. Circulation 2001;103(18):2236-41.]. These individual differences in the extent of theresponse to a particular inflammatory stimulus can have a genetic basis[Westendorp R G, Langermans J A, Huizinga T W, Verweij C L, Sturk A.Genetic influence on cytokine production in meningococcal disease.Lancet 1997; 349(9069):1912-3.]. Unfortunately, such heterogenicresponses limit the utility of downstream acute-phase reactants, suchas, for example, hsCRP as inflammatory marker for the riskstratification. In contrast to this, PlGF appears to be a directproximal stimulus for inflammatory processes within the vascular wall[Luttun A, Tjwa M, Moons L, et al. Revascularization of ischemic tissuesby PlGF treatment, and inhibition of tumour angiogenesis, arthritis andatherosclerosis by anti-Flt1. Nat Med 2002; 8(8):831-40.]. Indeed,elevated PlGF serum levels were extremely informative, specifically inpatients with acute coronary syndromes, but not non-elevated hsCRP serumlevels. In this cohort of patients, elevated PlGF serum levelsidentified a subgroup of patients with markedly elevated cardiac riskthat was similar to the high-risk patients that were defined by elevatedtroponin serum levels. Thus, elevated PlGF serum levels can indeedrepresent primary inflammatory indicators of coronary instability.

Furthermore, since the pro-inflammatory effects of PlGF can bespecifically inhibited by blocking its receptor Flt-1, these results canalso provide an approach for a novel anti-inflammatory therapeutictarget in patients with coronary arterial disease [Luttun A, Tjwa M,Canneliet P. Placental Growth Factor (PlGF) and Its Receptor Flt-1(VEGFR-I): Novel Therapeutic Targets for Angiogenic Disorders. Ann N YAcad Sci 2002; 979:80-93.]. The pro-inflammatory effects of PlGF can bespecifically inhibited by blocking its receptor Flt-1, and provide anovel anti-inflammatory therapeutic option in patients with coronaryarterial disease.

The results of the present study show that elevated blood levels of themetalloproteinase PAPP-A are associated with negative outcome inpatients with ACS. In agreement with an examination that was performedonly recently, the predictive value of PAPP-A plasma levels was mostprominent in patients without an increase of troponin. Thus, an elevatedPAPP-A plasma level is not only a marker of plaque-instability thatpromotes the development of ACS but, more important, indicates a badprognosis, before the occurrence of an acute ischemic event that iscaused by plaque-instability. In addition, elevated PAPP-A levelsprovide additional prognostic information, even in patients withelevated hsCRP plasma levels, suggesting that at least in some patientselevated hsCRP level are not associated with vascular inflammation. Theresult that the predictive values of PAPP-A were limited to patientswith low levels of the anti-inflammatory cytokine interleukin-10 furthersupports the concept that the balance between pro- and anti-inflammatorycytokines is [important] for the outcome for the patients in ACS. Usinga multivariate regression analysis, several biochemical markers,including troponin T, soluble CD40 ligand, interleukin-10, and PAPP-Awere identified as independent predictive markers for the outcome forthe patients during the subsequent six months of follow-up.

Cardiac troponines are sensitive and specific markers of myocardialnecrosis, secondary to thrombotic complications during an acute coronarysyndrome, and are highly predictive for the early clinical progressionafter the outbreak of ACS. However, the risk stratification in troponinnegative patients with acute coronary syndrome remains challenging.

About two thirds of the patients with ACS, but without elevations of theST-segment, exhibit normal values of Troponin, and more than half of thepatients exhibit inconclusive electrocardiographical results. During thefirst weeks following the outbreak of an acute coronary syndrome therisk of mortality or non-fatal myocardial infarction introponin-negative patients is at about 5 to 8%. Thus, the shortterm-occurrence of essential cardiovascular events remains relativelysubstantial in patients without evidences for myocardial necrosis,invoking the need for a further diagnostic processing. A continuousST-segment monitoring, stress tests, and perfusion imaging methods canbe of limited availability for the immediate risk stratification ofpatients from which it is assumed that they exhibit an acute coronarysyndrome. The present study shows that PAPP-A levels indicate a subgroupof patients without elevation of troponin that during the earlyprogression in time after the outbreak of symptoms exhibit anessentially higher risk for cardiac events (72 hours OR 3.17; 30 days:OR 3.33). In contrast to this, the patients without the modifications inthe ST-segment that were both negative for TnT and PAPP-A, were subjectto a very low risk (0.9% rate of occurrence). Thus, the determination ofPAPP-A in patients with ACS is an effective tool for the short term-riskstratification of patients without elevated troponin level.

The progression and the subsequent destabilisation of arterioscleroticplaques includes important changes in the structure of the arterialwall. The occurrence of a local state of inflammation in patients withACS is well established as determined by inflammatory markers, such as,for example, CRP. Metalloproteinases are also potential indicators ofarterial inflammation, and can contribute to the fragility of thelipid-rich arteriosclerotic plaques by degradation of extracellularmatrix, and eventually to its rupture. As described earlier for severalother metalloproteinases (MMP-1, MMP-3, MMP-12 or MMP-13) it was foundfor PAPP-A only very recently that it is expressed in eroded andreleased plaques, whereas the expression of PAPP-A could not be detectedin stable plaques. Other studies have also shown that patients withhyperechonic or isoechoinic carotid plaques exhibit significantlyelevated levels of PAPP-A than those with hypoechonic earlycarotid-lesions. The particular role of PAPP-A in the pathophysiology ofACS remains unclear. It was shown for PAPP-A that it is a specificactivator of the insulin-like growth factor-I (IGF-I), a potent mediatorof arteriosclerosis. As a matrix-metalloproteinase, PAPP-A could beinvolved in the processing of the extracellular matrix of plaques, and,consequently, affect the fibrous cap. This leads to a morphology of theplaque that is sensitive against erosion, rupture and subsequentthrombosis. The present study shows that that a single PAPP-Adetermination, obtained 8.7 hours after the occurrence of the symptomsprovides a significant predictive value for the occurrence of death andnon-fatal myocardial infarction during the following 6 months follow-up.These data suggest that PAPP-A plays an important pathophysiologicalrole in the destabilisation of the arteriosclerotic plaques during ACS.The production of PAPP-A by activated cells within the arterioscleroticlesions and its release into the extracellular matrix appears to betightly associated with the local inflammatory process that occurswithin the arterial wall as indicated by the significant positivecorrelation that was observed between CRP and PAPP-A levels. Indeed, thePAPP-A-levels were highly predictive in patients with elevated CRPlevels, whereas in patients with low CRP levels, PAPP-A did not serve asa significant predictor for the outcome of the patients (FIG. 26 a).Whilst CRP levels are connected with the troponin-increase, PAPP-Alevels appear to be less sensitive against smaller myocardial injury,which could be of particular importance in patients with ACS, out ofwhich at the time of arrival in the hospital about one third arepositive for troponin. In addition, PAPP-A levels neither interferedwith the predictive power of sCD40L, a marker of the activation ofplatelets in patients with ACS, nor did they affected these. Bymultivariate analysis PAPP-A, sCD40L and TnT all were found asindependent predictors of adverse outcome (Table 2). A combination ofPAPP-A and sCD40L was particularly obvious in patients that werenegative for TnT, suggesting that both markers reflect distinct signalpathways reflect that eventually contribute to a pro-inflammatory andpro-coagulating milieu in the coronary circulation. Supporting for acomplementary other than a competitive role in order to predict anadverse outcome in patients with ACS are the results of the inventorsthat the aggressive inhibition of the aggregation of the platelets byabciximab was particularly suitable in patients with elevated sCD40Llevels.

In summary, the results of the present invention show that elevatedplasma levels of PAPP-A as a marker of the vascular inflammation areassociated with an elevated risk for subsequent cardiac events. Thepredictive value of PAPP-A plasma levels was independent from elevatedtroponin levels that reflect the actual risk secondary to thromboticcomplications that lead to myocardial injury during an acute coronarysyndrome. Thus, an elevated PAPP-A plasma level is not only a marker ofthe plaque instability with respect to the progression to a myocardialinfarction, but also indicates a bad prognosis even after the occurrenceof an acute ischemic event that is caused by plaque instability.

In summary, it can be stated that the present study documents theimportant and independent role of PlGF as a marker for the diagnosticand therapeutic risk stratification. The elevated cardiac risk ofpatients with high PlGF-concentrations that received a standard therapywith heparin and aspirin was reversed by the glycoproteinIIb/IIIa-receptor antagonist abciximab. The combined use of troponinesand PlGF which both represent essential components of thepathophysiology in patients with acute coronary syndromes, providesimportant insights into the activity of the disease, the cardiologicrisk, and the effectiveness of a treatment by means of glycoproteinIIb/IIIa-inhibition using abciximab that is superior to the use of asingle marker.

In summary, the PlGF serum level represents an effective and reliablebiomarker of the vascular inflammation, and negative outcome in patientswith acute coronary syndromes. A determination of the PlGF serum levelssignificantly extends the predictive and prognostic information that isobtained from common inflammatory markers in acute coronary syndromes.

In the following, the invention shall now be further explained based onexamples with respect to the attached Figures, but without being limitedthereby. In the Figures:

FIG. 1 shows the association between serum concentrations of sCD40L andthe incidence of a cardiac event after 24 hours, 72 hours, 30 days, and6 months in the group of patients that received a placebo (n=544). Thepatients were grouped into 5 quintiles. The ranges of thesCD40L-concentrations were as follows: 0.003-1.9 μg/l (quintile 1),1.9-3.5 μg/l (quintile 2), 3.5-5.0 μg/l (quintile 3), 5.0-6.3 μg/l(quintile 4), and >6.3 μg/l (quintile 5). p<0.001 after 72 hours, 30days, and 6 months.

FIG. 2 shows a diagram according to Kaplan-Meier of the cumulativeincidence of myocardial infarction with lethal or non-lethal outcomeafter 72 hours (a), and 6 months (b) according to the base line-valuesof the sCD40L-concentration (diagnostic threshold value 5.0 μg/l) in theplacebo-group (n=544).

FIG. 3 shows the adapted risk quotient (including 95% confidenceinterval) in the treatment with abciximab according to thesCD40L-quintiles. A successful treatment is defined as a reduction oflethal or non-lethal myocardial infarction in the course of 6 months. Arisk quotient<1.0 indicates the successful treatment with abciximabcompared to placebo-treatment.

FIG. 4 shows a Kaplan-Meier-diagram of mortality and non-lethalmyocardial infarction 72 hours (a), and 6 months (b) according to thesCD40L-concentration in patients that either received a placebo orabciximab.

FIG. 5 shows the activation of platelets in dependency from the activityof the disease. The activity of platelets as determined bymonocyte-platelet-aggregates was significantly increased in patientswith stable coronary heart disease. An additional significant increaseof the activation of platelets was observed in patients with acutecoronary syndromes.

FIG. 6 shows that the sCD40L-serum concentrations do strictly correlatewith the extent of the activation of platelets in patients with andwithout acute coronary syndromes. The interrupted lines divide thepatients into tertiles according to the activation of platelets (<15%,15%-30%, >30%) or sCD40L-serum concentrations (<2.5 g/l; 2.5-4.5μg/l; >4.5 μg/l).

FIG. 7 shows the association between PlGF and hsCRP as a downstreamacute-phase reactant (n=1088).

FIG. 8 shows each of the PlGF and hsCRP serum levels according to thebase-line troponin T status (n=1088).

FIG. 9 shows the association between the serum concentrations of PlGFand the incidence of a cardiac event after 24 hours, 72 hours, 30 days,and 6 months in the group of patients that received a placebo (n=547).The ranges of the PlGF-concentrations were as follows: below andidentical to 13.3 ng/l (1. quintile), 13.4 to 19.2 ng/l (2. quintile),19.3 to 27.3 ng/l (3. quintile), 27.4 to 40.0 ng/l (4. quintile), andabove 40.0 ng/l (5. quintile). The differences in the rates of an eventbetween the quartiles were significant at 30 days (p 0.001), and 6months (p<0.001) of follow-up examination.

FIG. 10 shows receiver-operating-characteristic curve analysis for thepredictive value of PlGF serum levels for the occurrence of mortality ornon-lethal myocardial infarction at 6 months follow-up examination.

FIG. 11 shows a diagram according to Kaplan-Meier of the cumulativeincidence of myocardial infarction with lethal or non-lethal outcomeafter 72 hours (a) and 6 months (b) according to the base line-values ofthe PlGF-serum levels (diagnostic threshold value 27.0 ng/l; n=547).

FIG. 12 shows the predictive value of PlGF for the incidence ofmyocardial infarction with lethal or non-lethal outcome according tohsCRP serum levels (a), and troponin T serum levels (b). Diagnosticthreshold values were 27.0 ng/l for PlGF, 0.1 μg/l for troponin T, and10 mg/l for hsCRP; n=547).

FIG. 13 shows the predictive value of discharge-PlGF serum levels forthe patient-result at 6-months follow-up examination. Patients withelevated PlGF serum levels were subject to a higher cardiac risk, with arate of occurrence of 7.4%, compared to 2.2% for patients with PlGFserum levels below 27.0 ng/l (p=0.005).

FIG. 14 shows each of the PAPP-A and hsCRP-levels according to thebase-line troponin T status. Circles indicate outliers.

FIG. 15 shows soluble CD40 ligand and hsCRP-levels, respectively,according to the baseline PAPP-A status. Circles indicate outliers.

FIG. 16 shows the association between the PAPP-A plasma levels and therate of cardiac events at 24 h, 72 h, 30 days, and 6 months according tothe PAPP-A placebo group (n=547). The ranges of PAPP-A were as follows:(PAPP-A_(—)1)<4.5 mIU/l (n=111); (PAPP-A_(—)2) 4.5-7.5 mIU/l (n=108);(PAPP-A_(—)3) 7.6-12.6 mIU/l (n=109); (PAPP-A_(—)4) 12.7-24.0 mIU/l(n=110) and (PAPP-A_(—)5)>24.0 mIU/l. The in the rates of occurrencewere significant at 72 hours (p=0.019), 30 days (p=0.008), and 6 months(p=0.004).

FIG. 17 shows receiver-operating-characteristic curve analysis for thepredictive value of PAPP-A plasma levels for the occurrence of mortalityor non-lethal myocardial infarction at 6 months follow-up examination.

FIG. 18 shows Kaplan-Meier event rate curves that show the cumulativeoccurrence of death and non-fatal myocardial infarction at 72 hours (a),and 6 months (b) according to PAPP-A baseline plasma levels. Diagnosticthreshold value 12.6 mIU/l; n=547

FIG. 19 shows the predictive value of PAPP-A for the occurrence of deathand non-fatal myocardial infarction was limited to patients withelevated hsCRP levels (a), and patients with low levels of theanti-inflammatory cytokine IL-10 (b). Diagnostic threshold values 12.6mIU/1 for PAPP-A, 10 mg/l for hsCRP, and 3.5 ng/l for IL-10; n=547

FIG. 20 shows that the predictive value of PAPP-A for the occurrence ofdeath and non-fatal myocardial infarction was particularly informativein patients without troponin T increase (a). In patients that werenegative both for TnT and sCD40L, PAPP-A identified a subgroup thatsuffered from an elevated cardiovascular risk at 6 months follow-up (b).Diagnostic threshold values 12.6 mIU/1 for PAPP-A, 0.1 μg/l for TnT, and5.0 μg/l for sCD40L; n=547

EXAMPLES I. sCD40L

S1. Patients

Between Mai 1993 and December 1995, the CAPTURE-study registered 1265patients with refractory unstable angina (61% male, in the age of 61[48-72, 95% confidence interval]). All CAPTURE-patients were complainingabout reoccurring chest pain in the resting state, associated withECG-modifications during a treatment with intravenous heparin andglycerol trinitrate in an average of 14 hours. The completepatient-population was subjected to a coronary angiography before theoccurrence of a markedly coronary arterial disease with triggeringlesions of 17% that were suitable for an angioplasty was documented. Thepatients were randomly assigned to a treatment by abciximab or placebo.The treatment was started within 2 hours after the assignment. Coronaryinterventions were scheduled in all patients within 18 to 24 hours afterthe start of the treatment (CAPTURE. Lancet 1997; 349: 1429-35). Onaverage, blood samples (n=1096) were obtained (base line) 8.7 (3.6-11.3)hours after onset of the symptoms.

Primary endpoints of the study were mortality, myocardial infarction, orthe necessity for immediate intervention (angioplasty, bypass-surgery ofthe coronary artery) due to an instability during 30 days or 6 months.In patients that suffered from a heart attack during the period in thehospital, this was accordingly diagnosed, if their values of theenzymatic activity of the creatine kinase in at least two samples wasmore than three times as high than the upper limit of the normal range,or if their ECG exhibited novel distinct Q-waves in more than twosubsequent intervals. This strict definition was chosen in order toexclude any unimportant small increase of the creatine kinase afterPTCA. In patients with a myocardial infarction after discharge, this wasdefined accordingly, if their values of the enzymatic activity of thecreatine kinase were more than twice as high than the upper limit of thenormal range, or if their ECG exhibited novel distinct Q-waves in two ormore subsequent intervals. The secondary endpoint was the symptomaticcoronary restenosis of the treated lesion with a diameter of thestenosis of ≧70%, and the necessity for a repeated revascularisationduring the subsequent 6 months.

2. Validation of Patients with Acute Chest Pain

A separate group of 626 patients with chest pain (161 women and 465 men,average age 61 [38-82] years) that were presented in the emergency roomin a consecutive row with acute chest pain that lasted for less than 12hours (average 5.1 [2.1-10.4] hours). Patients with a characteristicST-elevation in the base-ECG or a documented acute myocardial infarctionduring the preceding 2 weeks were not taken into account. Blood sampleswere obtained at the time of admission (before the start of treatment),and 4 hours later, kept on ice, centrifuged within 20 minutes aftersample collection, and stored at −80° C. for later analysis. It wasfound that this treatment led to reproducible results during thedetermination of the sCD40L-concentrations (Nannizzi-Alaimo L,Rubenstein M H, Alves V L, Leong G Y, Phillips D R, Gold H K.Circulation 2002; 105:2849-54). The patients were observed untildischarge from the hospital and 30 days thereafter in order to registerlethal and non-lethal myocardial infarctions. The presence of a coronaryarterial disease was detected through one of the following criteria:ECG-indications of a myocardial ischemia (novel changes in theST-stretch or inversion of the T-wave), a coronary heart disease in theanamnesis (myocardial infarction or coronary revascularisation, apositive stress test or restriction of the diameter of a main coronaryartery by at least 50% in an early angiogram). Patients without coronaryheart disease had to show a normal coronary angiogram. The activation ofplatelets was detected by means of flow cytometry in a subgroup ofpatients including 131 patients with acute coronary syndromes, 20patients with stable coronary heart disease, and 10 patients withexcluded coronary heart disease.

3. Biochemical Analysis

Plasma samples, anti-coagulated with sodium heparin, were centrallystored at −80° C. The determination of the cardiac markers wereperformed in the research laboratory of the university Frankfurt withoutknowledge of the disease history of the patient and the treatment asordered. The plasma levels of sCD40L (detection limit 0.005 μg/l),soluble P-selectin (0.5 μg/l), tumour necrosis factor-α (TNF-α, highlysensitive; 0.12 ng/l), and soluble intracellular adhesion molecule-1(ICAM-1; 0.35 μg/l) were measured by means of ELISA (R&D systems,Wiesbaden). For a quantification of troponin T (TnT), a one-stepenzyme-immunoassay on the basis of the electrochemiluminescencetechnology (Elecsys 2010, Roche Diagnostics; detection limit 0.01 μg/l)was used. C-reactive protein (CRP) was measured with the aid of theBehring BN II nephelometer (Behring Diagnostics; detection limit 0.2μg/l).

5. Quantitative Determination of sCD40L

The sCD40L-concentration was determined by using thesandwich-enzyme-immunoassay-technology (R&D Systems, Wiesbaden). Amicrotitre plate was coated with a polyclonal antibody beingspecifically directed against sCD40L. Standards and samples werepipetted into the wells, and present sCD40L was bound by the immobilisedantibody. After unbound material was washed away, an enzyme-coupledpolyclonal antibody that was specifically directed against sCD40L wasadded to the wells. After a washing step in order to remove unboundantibody-enzyme-reagent, a substrate-solution was added to the wells,and the colour developed in relation to the amount of sCD40L that wasbound in the first step. The development of colour was stopped, and theintensity of the colour was measured.

6. Fast Assay for the Detection of CD40L

A fast assay on the basis of the chromatography-solid phase-technologywith a cocktail from gold-labelled polyclonal indicator-antibodies fromthe mouse and biotinylated polyclonal capture-antibodies was developed.The assay system contained at least 0.3 μg of each antibody. 200 μlheparinised whole blood or centrifuged plasma were added to the testdevice. After separation of the cellular blood components from theplasma fraction through a fibreglass-fleece, the plasma migratingthrough the fleece was taken up into a buffer, and added to the adsorbedantibodies. The antibodies and the sCD40L-molecules of the samplesformed sandwich-complexes that migrated to the signalling zone, andaccumulated in the vision panel by means of interaction withbiotin-streptavidin. Positive results (sCD40L 4.7 μg/l were indicated bya coloured line that developed within 15 minutes. The indicatorantibodies that were unbound migrated further, and were bound at acontrol line consisting of a solid phase-antimouse-IgG-antibody (≧0.2μg). The occurrence of said control line downstream from the signallingline confirmed the accurate test function including the unobstructedflow of the plasma.

7. In Vivo-Activation of Platelets

Blood samples that were anti-coagulated with sodium citrate wereimmediately treated for 10 minutes with 1.1% para-formaldehyde in PBS,diluted 4.6-fold in distilled water for a lysis of the erythrocytes, andthe fixed cells were washed in PBS. In order to determine P-selectin inplatelets the resuspended pellet was incubated 60 minutes withphycoerythrine (PE)-conjugated glycoprotein IIb-specific monoclonalantibody (CD41; Dako Carpenteria, California), and fluoresceinisothiocyanate (FITC)-conjugated P-selectin-specific monoclonal antibody(BD Pharmingen, San Diego, Calif.). The platelets were characterised bytheir characteristic forward- and sideward-light scattering, and thebinding of the PE-conjugated glycoprotein IIb-specific antibodies. Theactivation of platelets is expressed in % of the P-selectinpositiveplatelets. In order to identify circulatingmonocyte-platelet-aggregates, the cells were stained withFITC-conjugated glycoprotein IIIa-specific monoclonal antibodies (CD61;Dako) and PE-conjugated monoclonal antibodies against CD14 (BDPharmingen). The monocytes were identified by their characteristicforward- and sideward-light scattering properties and the binding ofPE-conjugated CD14-specific antibodies. Monocyte-platelet-aggregateswere defined as monocytes in which glycoprotein IIIa could be detected,and were indicated in percent of the total number of monocytes(Michelson A D, Barnard M R, Krueger L A, Valeri C R, Furman M I,Circulation 2001; 104:1533-7). Every staining was incubated in thepresence of saturating concentrations of a monoclonal non-conjugatedrat-antibody against Fc-receptor (anti-CD16/32, BD Pharmingen) in orderto reduce the unspecific binding, and isotypeidentical antibodies servedas control (IgG₁-PE and IgG_(2a)-FITC; BD Pharmingen). In total, 50,000signals were analysed with the aid of FACS-Calibur (Becton/Dickinson,Heidelberg), and the CellQuest-software (BD Pharmingen).

8. Statistical Methods

The assay results were compared with the data base after a blindevaluation of the biochemical markers and the activation of platelets.In order to be able to distinguish patients with different grades of acardiac risk, an orientated data analysis was chosen. CAPTURE-patientswere grouped according to the sCD40L-concentration of the quintile. Alogistic regression analysis was performed for each of the four pointsin time (24 hours, 72 hours, 30 days, and 6 months), and patients in thefirst quintile (sCD40L<2.0 μg/l) served as a reference. Receiveroperating characteristics (ROC) curve analysis over the dynamic range ofthe sCD40L-test was used in order to identify the thresholdconcentrations for sCD40L that provided the highest predictive valuesfor the risk stratification of patients with acute coronary syndromes.The effect of an increase of the biochemical marker on the fate of thepatient was evaluated by using a Cox proportional-hazards regressionmodel that included the base line-values of the prognostic factors (e.g.ECG-findings, cardiac risk factors, age and gender), and the randomlyselected treatment (Harrell F E, Jr., Lee K L, Pollock B G. J NatlCancer Inst 1988; 80:1198-202). All results for continuous variableswere expressed as medians with 95% confidence interval. In experimentswith more than two subgroups, identical intermediate groups wereanalysed by the T-test (two-sided) or ANOVA. Post hoc-range tests andpair-wise multiple comparisons were performed with the T-test(two-sided) with Bonferroni-adjustment. The comparison of categoricalvariables was performed with the Pearson-chi²-test. All analyses wereperformed with SPSS 11,0 (SPSS, Inc.). Values of p<0.05 were regarded asstatistically significant.

Example 1a Association Between Cardiac Risk and sCD40L-Concentration

The characteristics of the outcome for the population that was selectedfor this study (n=1088, 86% of the CAPTURE-patients) did not differ fromthe total population of the study regarding age, gender, cardiovascularrisk profile, and accompanying treatment before and after the randomselection. The reduction of cardiac events in the abciximab-group of thepopulation was comparable with the overall CAPTURE-population (beforePTCA: 2.2% placebo vs. 0.9% abciximab, p=0.094; after PTCA: 7.9% vs.3.5%, p=0.002; after 30 days: 9.0% vs. 4.2%, p=0.002) (CAPTURE. Lancet1997; 349: 1429-35).

sCD40L could be detected in the base line-serum samples of all 1088patients with an average of 4.5 μg/l (range 0.003-20.4). ThesCD40L-concentration did not correlate with the measured concentrationsof TnT (r=0.14) and CRP (r=0.11). The patients of the placebo-group(n=544) were grouped into quintiles in accordance with theirsCD40L-concentrations as measured: (sCD40L 1)<1.93 μg/l (n=100), (sCD40L2) 1.93-3.50 μg/l (n=102), (sCD40L 3) 3.50-5.00 μg/l (n=121), (sCD40L 4)5.00-6.30 μg/l (n=115) or (sCD40L 5)>6.30 μg/l (n=106). During the first24 hours, the combined endpoints mortality and non-deadly myocardialinfarction were slightly elevated only in the fifth sCD40L-quintilecompared to the first quintile (p=0.13) (FIG. 1). At later time points(72 hours, 30 days, 6 months), these events occurred significantly moreoften both in the fourth as well as in the fifth (p=0.003, p=0.0004 orp=0.001).

On the basis of the above described results, the samples of the patientswere grouped in accordance with the calculated threshold concentrations.221 patients (40.6%) exhibited sCD40L-concentrations of 5.0 μg/l orabove, and 323 patients exhibited values of <5.0 μg/l. As shown in Table1, no significant differences occurred in the characteristics of theoutcome of both groups. In patients with lower sCD40L-concentrations thecombined endpoints lethal and non-lethal myocardial infarction weresignificantly different from those of patients with elevatedsCD40L-concentrations both 24 hours before the method (4.1% vs. 0.9%;p=0.016) as well as 72 hours thereafter (including coronaryinterventions in all patients) (13.1% vs. 4.3%; vs. 4.3%; p<0.001) (FIG.2 a). During the 6-month follow-up period the curves that indicated thefrequency of an event in patients with high or low sCD40L-concentrations(FIG. 2 b) diverged further. Significant differences were found bothafter 30 days (14.5% vs. 5.3%; p<0.001) as well as after 6 months (18.6%vs. 7.1%; p<0.001). Due to the relatively low mortality of theCAPTURE-group, the endpoint mortality after 6 months did notsignificantly differ between both groups (2.3% vs. 1.5%; p=0.72). Thepredictive value of sCD40L was independent from a myocardial necrosis. Agroup of patients with elevated cardiac risk (13.6%) was identified inTnT-negative patients by means of the sCD40L-concentration that did notdiffer significantly from the cardiac risk of TnT-positive patients(14.0%; p=1.00). Receiver operating characteristics curve analysisconfirmed a threshold concentration of 5.0 μg/l for the maximisedpredictive value of sCD40L. Repeated non-urgent cardiologicinterventions during the first 6 months in patients with highsCD40-concentrations were not significantly different from those inpatients with lower sCD40L-concentrations (6.2% vs. 4.5%; p=0.45).

Out of 626 patients without ST-elevation that were admitted in theemergency room with acute chest pain, 308 patients suffered from anacute coronary syndrome (117 patients had suffered from an acutemyocardial infarction, supported by a troponin-increase 0.1 μg/l). Thefollowing diagnosis was made in the other patients: n=91 stable angina,n=10 lung embolism, n=11 congestive heart insufficiency, n=7myocarditis, and n=199 no evidence for a heart disease.sCD40L-concentrations were significantly higher in patients with acutecoronary syndromes (4.53 [3.19-5.87]μg/l) compared to patients withstable angina (2.41 [1.99-3.52] μg/l; p<0.001) or patients withoutindications of a heart disease (1.57 [0.88-1.76] μg/l; p<0.001). Theaverage value for the upper reference limit (URL) of 97.5 in patientswithout indications of cardiac disturbances was 4.7 μg/l, and theURL-average value of 99 was found at 6.2 μg/ml. Similarly to the resultsthat were obtained in the CAPTURE-study, the sCD40L-serum concentrationsdid not correlate with necrosis markers (troponin T), inflammatorymarkers (CRP, TNF-α), and adhesion molecules (ICAM-1). In patients withacute coronary syndromes, 43.5% exhibited sCD40L-serum concentrationsabove the 97.5 URL-average value, and 21.8% exhibited sCD40L-serumconcentrations above des 99 URL-average value. By using a definedthreshold concentration for sCD40L of 5.0 μg/l, patients with anelevated sCD40L-serum concentration were identified as ahigh-risk-population (defined risk quotient 3.00 [1.35-6.71]; p=0.009).Inside the overall heterogenic population of patients with chest pain,the defined threshold concentration of 5.0 μg/l was also reliable inidentifying patients that exhibited a profound risk for cardiologicevents during the following 30 days (defined risk quotient 6.65 [3.18 to13.89]; p<0.001). The area under the ROC-curve was 0.75 [0.67-0.83], anda maximised predictive value was reached at a threshold concentration of4.8 μg/l.

Example 2a Angiographic Results and sCD40L-Concentration

The base line coronary angiograms in patients with elevatedsCD40L-concentrations showed more complex characteristics of thelesions. Lesions of type B2+ or C were documented in 40.6% of thepatients with high sCD40L-concentrations, whereas only 27.5% of thepatients with low sCD40L-concentrations exhibited more complexcharacteristics of the lesions (p=0.004).

In 59.6% of the patients with high sCD40L-concentrations and in 58.9% ofthe patients with low sCD40L-concentrations the base line value for theTIMI-flow-through was normal (p=0.049). For 7.8% of the patients withhigh sCD40L-concentrations a TIMI-flow-through=1 was documented comparedto 5.7% of the patients with low sCD40L-concentrations (p=0.67).

A thrombus at the time of presentation was visible in 11.1% of thepatients with high sCD40L-concentrations compared to 4.8% patients withlow sCD40L-concentrations (p=0.009). All patients with visible thrombusformation exhibited sCD40L-concentrations of >2.5 μg/l.

Example 3a Effect of Abciximab on the sCD40L-Concentration

A logistic regression analysis pointed towards a significant associationbetween the effectiveness of a treatment with abciximab and thesCD40L-concentrations (p<0.001). The patients were grouped intoquintiles as noted above. For the first two quintiles, no differenceswere observed with respect to a cardiac risk at treatment with placeboor abciximab (FIG. 3). A significant and comparably pronounced reductionof cardiac events was documented for the upper two quintiles. Thefinding of a change of the disparity to 0.35 between the second and thequintiles suggests a favourable threshold concentration of thesCD40L-concentrations in this range. Accordingly, the curves that showthe frequency of a cardiac event in form of a lethal or non-lethalmyocardial infarction were generated by using a threshold concentrationof 5.0 μg/l. Events before these methods were rare in patients with lowsCD40L-concentrations that received placebo (0.9%), whereas in 3.4% ofthe patients events occurred in association with a coronary intervention(FIG. 4 a). In patients with low sCD40L-concentrations no difference wasobserved between patients that received abciximab and those thatreceived placebo (24 hours: 1.2% vs. 0.9%; 72 hours: 3.8% vs. 4.3%). Inthe following 6 months, only very few additional events were registered.The overall frequency was 7.1% (FIG. 4 b).

In contrast to this, the frequency of attacks was significantly higherin patients with high sCD40L-concentrations receiving placebo, bothbefore PTCA (4.1%) as well as under PTCA (9.0%) as well as in thefollowing time after discharge, leading to an overall frequency of 18.6%after six months. Events that occurred before and under PTCA wereeffectively reduced to 0.5% before PTCA (risk quotient 0.12 [0.01-0.92];p=0.013), and 2.9% for PTCA-related events (risk quotient 0.19[0.08-0.49]; p<0.001) by a treatment with abciximab. This improvementwas maintained for six months after the event, leading to a cumulativefrequency of the events of 7.8% vs. 18.6% in the placebo group (riskquotient 0.37 [0.20-0.68]; p=0.001). This value is comparable with theone that was observed in patients with low sCD40L-concentrations (7.1%).Furthermore, the effect of the glycoprotein IIb/IIIa-inhibition withoutthe occurrence of a myocardial necrosis was obvious. In TnT-negativepatients, a subgroup of patients was identified by means of thesCD40L-concentration that exhibited a significant reduction of cardiacevents when they received abciximab (2.8% vs. 10.2%; placebo vs.abciximab; p=0.022).

The dissolution of a thrombus during the ordered treatment beforecoronary intervention was particularly noticeable in patients with highsCD40L-concentrations when treated with abciximab, with a relativereduction of 63% (placebo: −21%; p<0.01). A thrombus formation inpatients with low sCD40L-concentrations was rare and a significantdissolution was neither achieved in the placebo group (p=0.75) nor inthe abciximab-group (p=0.82).

Example 4a sCD40L as Marker of an Activation of Platelets In Vivo

In order to verify the hypothesis that sCD40L indeed constitutes amarker for the activation of platelets, the ratio of the activation ofplatelets and the sCD40L-serum concentrations was prospectively analysedin a subgroup of patients with acute chest pain (n=151). The extent ofthe activation of platelets in patients with acute coronary syndromeswas significantly elevated compared both to patients with stablecoronary heart disease as well as to patients without coronary heartdisease (FIG. 5). A strict correlation between the activation ofplatelets as determined by means of monocyte-platelet-aggregates (%) andthe sCD40L-concentration was observed (r=0.75; p<0.0001) (FIG. 6).Similar results were obtained for P-selectin (p<0.001). The patientswere grouped into tertiles according to their measuredsCD40L-concentrations: (sCD40L1)<2.5 μg/l (n=55), (sCD40L 2) 2.5-4.5μg/l (n=50), and (sCD40L 3)>4.5 (n=56), respectively. In patients of thefirst sCD40L-tertile, the percentage of monocyte-platelet-aggregates was11.3% [9.6-12.9]. In the second and third sCD40L-tertiles the activationof platelets was significantly higher (22.3 [19.8-24.8]%, and 34.1[30.0-38.3]%; p<0.001, respectively). It is particularly interestingthat in all patients (with the exclusion of one patient) that exhibitedsCD40L-serum concentrations of >4.5 μg/l, at least 15% of the monocyteswere aggregated with platelets, indicating a pronounced activation ofplatelets in these patients. In contrast thereto the serumconcentrations of soluble P-selectin showed a markedly less pronouncedassociation with the activation of platelets (r=0.41 formonocyte-platelet-aggregates, and r=0.36 for P-selectin, respectively).It could be found in a multi-variance-regression analysis that adiabetes-disease (p=0.015), a hypercholesterolemia (p=0.006), and thesCD40L-concentration (p<0.0001), but not the concentration of solubleP-selectin, was significantly associated with the activation ofplatelets (monocyte-platelet-aggregates>30%).

TABLE 1 Base line characteristics of sCD40L in the placebo group sCD40Llow sCD40L high n 323 221 Male 73.3% 70.2% Age 60.5 ± 9.9 62.3 ± 10.5Anamnesis Angina for more than 4 weeks 57.5% 54.6% infarction 14-30 days 3.5%  2.4% infarction more than 30 days 19.5% 21.0% PTCA 19.1% 15.9%CABG  3.5%  3.4% Risk factors Diabetes 12.1%  9.6% Hypercholesterolemia32.5% 33.6% Hypertension 39.3% 34.5% Smoker 40.9% 42.1% Medicationbefore registration Aspirin 98.1% 97.7% Heparin i.v. 99.2% 98.6% Nitratei.v. 98.8% 99.7% Beta-blocker 65.0% 61.4% Ca²⁺-antagonists 56.0% 55.2%

TABLE 2 Lethal and non-lethal myocardial infarction within the first 6months (multi-variance-regression analysis) Variable OR 95% CI p-valueGender 0.91 0.68-1.39 0.16 Age higher than 65 years 1.36 0.91-1.82 0.34Diabetes mellitus 1.22 0.83-1.49 0.61 Hypercholesterolemia 0.900.68-1.13 0.59 Hypertension 1.00 0.89-1.04 1.00 History of a CHD 0.860.65-1.19 0.72 ST-depression 1.04 0.76-1.54 0.74 TnT > 0.1 μg/l 2.941.75-7.26 <0.001 CRP > 10 mg/l 2.03 1.11-3.59 0.018 sCD40L > 5.0 μg/l2.71 1.51-5.35 0.001

II. PlGF

PlGF was initially identified in the placenta and stimulates vascularsmooth muscle growth, recruits macrophages in arteriosclerotic lesions,up-regulates the production of TNF-α, and MCP-1 by macrophages, andstimulates the pathological angiogenesis. Much more important, it wasexperimentally shown from the inhibition of the effects of PlGF byblocking its receptor tyrosine kinase Flt-1, that this represses boththe growth of arteriosclerotic plaques as well as the vulnerability bythe inhibition of the inflammatory infiltration of cells. These datasuggest that PlGF might serves as a primary inflammatory indicator ofthe instability of arteriosclerotic plaques.

Thus, the prognostic significance of PlGF in patients with acutecoronary syndromes was employed by using the data of the patients withacute coronary syndromes that were included in the CAPTURE study (c7E3“Anti Platelet Therapy in Unstable Refractory angina”), and thediagnostic and the prognostic significance was then preliminaryvalidated in a large population of patients that were admitted with painin the chest. The PlGF serum levels were measured in 1088 patients fromthe CAPTURE study with acute coronary syndromes. Furthermore, thediagnostic and prognostic significance of PlGF serum levels waspreliminary validated in a heterogeneous group of 619 patients withacute pain in the chest. The incidence of myocardial infarction withlethal or non-lethal outcome was recorded during the follow-up period.

1. Patients

Design of the sets of patients with acute coronary syndromes. TheCAPTURE-study registered 1265 patients with acute coronary syndromes(61% male, in the age of 61±10 years). All CAPTURE-patients werecomplaining about reoccurring chest pain in the resting state,associated with ECG-modifications during a treatment with intravenousheparin and glycerol trinitrate. The overall patient-population wassubjected to a coronary angiography before the randomisation whichsignificantly indicated the occurrence of a markedly coronary arterialdisease with triggering lesions of >70% that were suitable for theangioplasty. Heparin was applied starting before the randomisation to atleast 1 h after the PTCA procedure. Coronary interventions werescheduled in all patients within 18 to 24 hours after the start of thetreatment. The patients were randomly assigned to a treatment byabciximab or placebo. Primary endpoints of the study were mortality andnon-fatal myocardial infarction during the 6-months follow-up-period.Serum samples were taken 8.7 [75% CI 3.6-11.3] hours after onset of thesymptoms.

2. Validation of Patients with Acute Chest Pain

A separate group of 626 patients with chest pain (161 women and 465 men,average age 61 [38-82] years) that were presented in the emergency roomin a consecutive row with acute chest pain that lasted for less than 12hours (average 5.1 [2.1-10.4] hours) was established as a set. Patientswith a characteristic ST-elevation in the base-ECG or a documented acutemyocardial infarction during the preceding 2 weeks were not taken intoaccount. Serum samples were obtained at the time of admission (beforethe start of treatment), and 4 hours later, kept on ice, centrifugedwithin 20 minutes after sample collection, and stored at −80° C. forlater analysis. The patients were observed until discharge from thehospital and 30 days thereafter, in order to register lethal andnon-lethal myocardial infarctions. The presence of a coronary arterialdisease was detected by one of the following criteria: ECG-indicationsof a myocardial ischemia (novel changes in the ST-stretch or inversionof the T-wave), a coronary heart disease in the anamnesis (myocardialinfarction or coronary revascularisation, a positive stress test orrestriction of the diameter of a main coronary artery by at least 50% inan early angiogram). Patients without coronary heart disease had to showa normal coronary angiogram.

Primary endpoints of the study were mortality, myocardial infarction orthe necessity for immediate intervention (angioplasty, bypass-operationsof the coronary artery) due to an instability during 30 days or 6months. In patients that suffered from a heart attack during the periodin the hospital, this was accordingly diagnosed, if their values of theenzymatic activity of the creatine kinase in at least two samples wasmore than three times as high than the upper limit of the normal range,or if their ECG exhibited novel distinct Q-waves in more than twosubsequent intervals. This strict definition was chosen in order toexclude any unimportant small increase of the creatine kinase afterPTCA. In patients with a myocardial infarction after discharge, this wasdefined accordingly, if their values of the enzymatic activity of thecreatine kinase were more than twice as high than the upper limit of thenormal range, or if their ECG exhibited novel distinct Q-waves in two ormore subsequent intervals. The secondary endpoint was the symptomaticcoronary restenosis of the treated lesion with a diameter of thestenosis of 70%, and the necessity for a repeated revascularisationduring the subsequent 6 months.

3. Biochemical Analysis

Serum samples were centrally stored at −80° C. The determinations of thecardiac markers were performed in the research laboratory of theuniversity Frankfurt without knowledge of the disease history of thepatient and the treatment as ordered. The serum levels of PlGF and VEGFwere measured by means of ELISA (R&D Systems, Wiesbaden). For aquantification of cardiac troponin T (TnT), a one-stepenzyme-immunoassay on the basis of the electrochemiluminescencetechnology (Elecsys 2010, Roche Diagnostics) was used. Highly sensitiveC-reactive protein (hsCRP) was measured with the aid of the Behring BNII nephelometer (Behring Diagnostics). A diagnostic threshold value of10.0 mg/l was used [9, 18].

4. Quantitative Determination of PlGF

The PlGF-concentration was determined by using thesandwich-enzyme-immunoassay-technology (R&D Systems, Wiesbaden). Amicrotitre plate was coated with a polyclonal antibody that wasspecifically directed against PlGF. Standards and samples were pipettedinto the wells, and present PlGF was bound by the immobilised antibody.After unbound material was washed away, an enzyme-coupled polyclonalantibody which was specifically directed against PlGF was added to thewells. After a washing step in order to remove unboundantibody-enzyme-reagent a substrate-solution was added to the wells, andthe colour developed in relation to the amount of PlGF that was bound inthe first step. The development of colour was stopped, and the intensityof the colour was measured.

5. Statistical Methods

The assay results were compared with the data base after a blindevaluation of the biochemical markers. In order to be able todistinguish patients with different grades of a cardiac risk, anorientated data analysis was chosen. The Cox proportional-hazardsregression model was used in order to estimate the relative risk forcardiovascular events, and the patients were grouped according to thePlGF-concentration of the quintiles. The post-hoc analysis of thequintiles was performed by using the Cox proportional-hazards regressionmodel with the PlGF quintiles as a categorical variable, and patients inthe first quintile served as a reference. Receiver operatingcharacteristics (ROC) curve analysis over the dynamic range of thePlGF-assay was used in order to identify the threshold concentration forPlGF that provided the highest predictive value for the riskstratification of patients with acute coronary syndromes. The effect ofthe baseline characteristics (whereby p=0.10 was required in order toinclude a variable into the model) and other biochemical markers on anyassociations between PlGF levels and cardiovascular events as observedwas performed by using stepwise Cox proportional-hazards regressionmodel. All results of the continuous variables are expressed asmedian±standard deviation. The comparison between the groups wasanalysed by a t-test (two-sided). The comparison of categoricalvariables was generated by the Pearson χ² test. p-values of <0.05 wereregarded as statistically significant. All analyses were performed withSPSS 11.0 (SPSS Inc., Chicago).

Example 1b Association Between Cardiac Risk and PlGF-Concentration

The characteristics of the outcome for the population that was selectedfor this study (n=1088, 86% of the CAPTURE-patients) did not differ fromthe total population of the study in view of age, gender, cardiovascularrisk profile, and accompanying treatment before and after the randomselection. The reduction of cardiac events in the abciximab-group of thepopulation was comparable with the overall CAPTURE-population, bothbefore PTCA (2.2% placebo versus 0.9% abciximab; p=0.07), as well asafter PTCA (7.9% versus 3.5%; p=0.001), and at 30 days (9.0% versus4.2%; p=0.001).

PlGF could be detected in the base line-serum samples of 95.6% of thepatients of the study with an average of 23.0 ng/l (range 7.0-181.2).The PlGF serum level did not correlate with the measured concentrationsof troponin T (r=0.14) and VEGF levels (r=0.07), but exhibited asignificant correlation with hsCRP serum level (r=0.48) (FIG. 7). ThePlGF serum level did not differ between troponin T-positive and troponinT-negative patients, whereas the hsCRP serum levels were significantlyhigher in troponin T-positive patients (FIG. 8). The patients of theplacebo-group (n=547) were grouped into quintiles according to theirmeasured PlGF serum levels: (PlGF 1)<13.3 ng/ln=109), (PlGF 2) 13.4-19.2ng/l (n=110), (PlGF 3) 19.3-27.3 ng/l (n=110), (PlGF 4) 27.3-40.0 ng/L(n=109), and (PlGF 5)>40.0 ng/l (n=109), respectively. During the first24 hours the combined endpoints mortality and non-lethal myocardialinfarction were not different between the PlGF-quintiles (p=0.11) (FIG.9). At later points in time (72 hours, 30 days, 6 months) theevent-rates showed significant differences between the PlGF quintiles.At 72 hours follow-up examination the event-rates were significantlyhigher in the fourth and fifth quintile when compared with the firstquintile (p=0.038 and p=0.011, respectively). During the subsequent 6months follow-up examination, the event-rates diverged further, leadingto significant differences for the fourth and fifth quintile at 30 days(p=0.005 and p=0.017, respectively), and 6 months follow-up examination(p=0.002 and p=0.001, respectively).

Receiver operating characteristics curve analysis confirmed a thresholdvalue of 27.0 ng/l for the maximised predictive value of PlGF (FIG. 10).On the basis of this threshold value, 223 patients (40.8%) had PlGFserum levels above or identical to 27.0 ng/l, and 324 patients below27.0 ng/l. As shown in Table 3 small differences in the outcomecharacteristics of both groups occurred. Patients with elevated PlGFserum levels more often were diabetics and had hypertension andexhibited significantly higher hsCRP serum levels (Table 3). In patientswith high PlGF serum level, the combined endpoints lethal or non-lethalmyocardial infarction were significant different from patients with lowPlGF serum levels. After 72 hours (including coronary intervention inall patients) 12.1% of the patients with high PlGF serum levelsexperienced a negative event, compared to 4.9% for patients with lowPlGF serum level (p=0.002) (FIG. 11 a). During the 6-month follow-upperiod the curves indicating the frequency of an event diverged furtherbetween patients with high or low PlGF serum levels (FIG. 11 b).Significant differences were found both after 30 days (15.8% versus3.6%; p=0.001) as well as after 6 months (20.3% versus 4.9%; p<0.001).Despite the relatively low mortality of the CAPTURE-group theendpoint-mortality between both groups (4.0% versus 0.9%; p=0.021)differed significantly after 6 months. In a multivariate analysis thatincluded the baseline characteristics and biochemical markers (troponinT, VEGF, hsCRP), PlGF remained as an independently effective predictorof elevated cardiac risk both at 30 days follow-up examination (adjustedhazards-ratio 3.34 [95% CI 1.79-6.24]; p<0.001), and at 6 monthsfollow-up examination (adjusted hazards-ratio 3.58 [95% CI 1.48-7.72];p<0.001) (Table 4). A separation of the patients into four groups basedon their PlGF and hsCRP levels showed that PlGF identified a subgroup ofpatients with low hsCRP serum levels that suffered from a significantlyelevated cardiac risk. Patients with low hsCRP serum levels but PlGFserum levels above 27.0 ng/l had a significantly higher risk compared topatients that exhibited low levels for both hsCRP and PlGF (23.6% versus3.9%; p=0.001) (FIG. 12 a).

Furthermore, the predictive value of PlGF was independent frommyocardial necrosis. High PlGF serum levels showed an elevated cardiacrisk both in troponin T-positive patients (15.4% versus 4.1%; P=0.005)as well as in troponin T-negative patients (26.1% versus 10.1%; p=0.001)(FIG. 12 b).

Example 2b Effect of Abciximab in View of the PlGF-Serum Level

A logistic regression analysis pointed towards a borderline-significantconnection between the effectiveness of a treatment with abciximab andthe PlGF-concentrations (p=0.043). Patients with elevated PlGF serumlevels that received abciximab had a significantly lower risk at 30 daysfollow-up examination (adjusted hazards-ratio 0.38 [0.19-0.74];p=0.005). This significant difference was maintained at 6 monthsfollow-up examination (0.57 [0.34-0.96]; p=0.037). In contrast to this,patients with low PlGF serum levels did not gain a significanttherapeutic benefit from a treatment with abciximab (30 days follow-upexamination:adjusted hazards-ratio 0.59 [0.27-1.33]; p=0.23).

Example 3b Discharge PlGF Serum Level are Predictive for the Long-TermOutcome

A second blood sample that was taken before discharge (7.2±4.5 daysfollowing randomisation) could be obtained for 489 patients of the 547placebo-patients (89.4%). The PlGF serum levels decreased from anaverage of 27.12±19.56 ng/l at baseline to 23.4±26.2 ng/l at discharge(p=0.012). For patients with a PlGF serum level at discharge of above27.0 ng/l, the occurrence of mortality and non-fatal myocardialinfarction was significantly higher when compared to patients having lowPlGF serum levels, both at 30 days (4.6% versus 0.8%; p=0.019) and 6months follow-up examination (7.4% versus 2.2%; p=0.005) (FIG. 13).

Example 4b Validation of the PlGF-Threshold Values in Patients withAcute Chest Pain

Out of 626 patients with acute chest pain, 308 patients suffered from anacute coronary syndrome (117 patients had a non-ST-elevated myocardialinfarction). The other patients were grouped according to the followingdiagnoses: n=91 stable angina, n=10 pulmonary embolism, n=11 congestiveheart failure, n=7 myocarditis, and n=199 no indications for a heartdisease. The PlGF serum level were significantly increased in patientswith acute coronary syndromes (average 28.3 [95% CI 21.3-2.2] ng/l) whencompared each to patients with stable angina (average 16.2 [95% CI13.8-18.6 ng/l; p=0.001), and patients without indications for a heartdisease (average 9.6 [95% CI 10.4-12.9 ng/l; p=0.001). The PlGF serumlevels in patients with non-ST-elevated myocardial infarction did notdiffer significantly from PlGF serum levels in patients with unstableangina (30.5 [95% CI 26.9-34.1] versus 28.3 [95% CI 21.3-32.2] ng/l;p=0.42). The 97.5 percent upper reference limit in patients withoutindications for a heart disease was 24.9 ng/l and the 99 percent upperreference limit was 27.3 ng/l. The PlGF serum level did not correlatewith markers of necrosis (troponin T [r=0.07]), but correlatedsignificantly with inflammatory markers (C-reactive protein [r=0.43]).

In patients with acute coronary syndromes, 44.8% of the patientsexhibited PlGF serum level above the 99 percent upper reference limit.By using the threshold values for PlGF of 27.0 ng/l patients withelevated PlGF serum level were underlying a significantly higher riskfor death and myocardial infarction (adjusted hazards-ratio 2.97 [95% CI1.74 to 9.06; p=0.014). Within the overall heterogeneous population ofpatients with chest pain the threshold value of 27.0 ng/l also reliablyidentified patients that were subject to the highest risk for death andmyocardial infarction (adjusted hazards-ratio 4.95 [95% CI 2.50 to 9.79;P 0.001).

Summary of the Examples 1b to 4b

In patients with ACS, PlGF does not correlate with VEGF, troponin T, andST-segment modifications, but exhibited a significant correlation withhsCRP (p=0.001). Patients with elevated PlGF serum level (>27.0 ng/l;40.8%) experienced a drastically elevated cardiac risk (death andnon-fatale myocardial infarction) both at 30 days (adjustedhazards-ratio 3.34 [95% CI 1.79-6.24]; p=0.001), and at 6 months(adjusted hazards-ratio 3.58 [95% CI 1.48-7.72]; p=0.001) after ACS. ThePlGF serum levels were specifically informative in patients with lowhsCRP levels. The preliminary validation in patients with acute chestpain led to the result that PlGF serum levels>27 ng/l reliablyidentified those patients being subject to the highest risk for deathand myocardial infarction (adjusted hazards-ratio 4.95 [95% CI 2.50 to9.79; p=0.001). The elevated risk in patients with higher PlGF serumlevels was reduced by the treatment with the glycoprotein receptorinhibitor abciximab (adjusted hazards-ratio 0.38 [0.19-0.74]; p=0.005).

TABLE 3 Base line characteristics according to the PlGF status for theplacebo group of the CAPTURE study (n = 547) PlGF low PlGF high p-valuen 324 223 Male 71.4% 69.2% 0.34 Age 61.4 ± 10.5 62.3 ± 10.5 0.32troponin T ≧ 0.1 μg/l 33.8% 40.4% 0.12 CRP ≧ 10.0 μg/l 29.1% 67.7%<0.001 ST-segment depression 46.0% 52.1% 0.18 T-wave inversion 51.4%52.1% 0.93 History of angina > 4 weeks 55.3% 57.4% 0.64 infarction < 30days 12.5% 13.6% 0.84 infarction > 30 days 20.3% 20.4% 0.97 PTCA 16.5%18.4% 0.56 CABG  3.2%  3.7% 0.88 Risk factors Diabetes  8.2% 12.5% 0.034Hypertension 33.4% 39.9% 0.019 Acute smoker 39.6% 41.8% 0.48 Medicationbefore registration Aspirin 97.9% 98.1% 1.00 Heparin i.v. 99.0% 98.9%0.98 Nitrates i.v. 99.4% 99.3% 1.00 Beta-blockers 63.5% 62.9% 0.91

TABLE 4 Multivariate Cox proportional-hazards-regression model forlethal and non-lethal myocardial infarction within the first 6 months offollow-up examination, derived from the placebo group of the CAPTUREstudy adjusted hazards- Variable ratio 95% CI p-value Gender 0.95 0.72to 1.68 0.38 Age > 65 years 1.22 0.65 to 1.47 0.50 Diabetes mellitus1.22 0.83 to 1.49 0.61 Hypercholesterolemia 0.90 0.68 to 1.13 0.59 Acutesmoker 0.66 0.42 to 1.25 0.18 Hypertension 1.04 0.91 to 1.25 0.95History of a coronary 0.86 0.65 to 1.19 0.72 revascularisationST-depression 0.96 0.55 to 1.42 0.81 hsCRP > 10.0 mg/l 0.95 0.62 to 1.570.88 troponin T > 0.1 μg/l 1.76 0.98 to 3.46 0.084 VEGF > 300 ng/l 2.161.05 to 4.11 0.031 PlGF > 27.0 ng/l 3.58 1.48 to 7.72 <0.001

III. PAPP-A and Combinations

The prognostic significance of PAPP-A in patients with acute coronarysyndromes was employed by using the data of the patients with acutecoronary syndromes that were included in the CAPTURE study (c7E3 “AntiPlatelet Therapy in Unstable Refractory angina”), and the diagnostic andprognostic significance in a large population of patients that wereadmitted with pain in the chest was then preliminary validated. ThePAPP-A serum levels were measured in patients with acute coronarysyndromes from the CAPTURE study. The incidence of myocardial infarctionwith lethal or non-lethal outcome was recorded during the follow-upperiod.

1. Patients

Design of the sets of patients with acute coronary syndromes. TheCAPTURE-study registered 1265 patients with acute coronary syndromes(61% male, in the age of 61±10 years). All CAPTURE-patients werecomplaining about reoccurring chest pain in the resting state,associated with ECG-modifications during a treatment with intravenousheparin and glycerol trinitrate. The overall patient-population wassubjected to a coronary angiography before the randomisation, whichsignificantly indicated the occurrence of a markedly coronary arterialdisease with triggering lesions of >70% that were suitable for theangioplasty. Heparin was applied starting before the randomisation to atleast 1 h after the PTCA procedure. Coronary interventions werescheduled in all patients within 18 to 24 hours after the onset of thetreatment. The patients were randomly assigned to a treatment byabciximab or placebo. Primary endpoints of the study were mortality andnon-fatal myocardial infarction during the 6-months follow-up-period.Myocardial infarction during the stay in hospital including theangioplasty methods were defined by values of the CK enzymatic activityof more than threefold the upper limit of normal in at least twosamples, and/or new significant Q-waves in two or more continuous leads.Myocardial infarction after discharge was defined as values of the CKenzymatic activity of more than twofold the upper limit of normal in atleast two samples, and/or new significant Q-waves in two or morecontinuous leads. Since it was shown for other markers, such as, forexample, troponin T (TnT) and soluble CD40 ligand (sCD40L) that theseinterfere with the treatment effect of the glycoprotein IIb/IIIareceptor antagonist abciximab the present analysis was limited toplacebo patients with available blood samples (n=547; 86% of the placebopatients). Blood samples were collected 8.7±4.9 hours after outbreak ofthe symptoms.

2. Biochemical Analysis

Serum samples were centrally stored at −80° C. The determinations of thecardiac markers were performed in the research laboratory of theuniversity Frankfurt without knowledge of the diseases history of thepatient and the treatment as ordered. The serum levels of PlGF and VEGFwere measured by means of ELISA (R&D Systems, Wiesbaden). For aquantification of cardiac troponin T (TnT), a one-stepenzyme-immunoassay on the basis of the electrochemiluminescencetechnology (Elecsys 2010, Roche Diagnostics) was used. Highly sensitiveC-reactive protein (hsCRP) was measured with the aid of the Behring BNII nephelometer (Behring Diagnostics). A diagnostic threshold value of10.0 mg/l was used. Highly sensitive interleukin-10 (IL-10), vascularendothelial growth factor (VEGF), and sCD40L were measured by means ofELISA (both R&D Systems, Wiesbaden, Germany). We used the following,earlier established diagnostic threshold values: 5.0 μg/l for sCD40L,300 ng/l for VEGF, 3.5 ng/l for IL-10. Cardiac PAPP-A was determined byusing a one-step enzyme-immunoassay on the basis of theelectro-chemiluminescence technology (Elecsys 2010, Roche Diagnostics,Mannheim, Germany). By using internal controls, the overall inaccuracyfor PAPP-A over the 8-week period was 8.5%.

3. Statistical Methods

The patients were grouped according to the PAPP-A plasma concentrationsof the quintiles. For each point in time (24 h, 72 h, 30 days, and 6months), the logistic regression model was used in order to determinethe relative risk for death and myocardial infarction. The effect of thebaseline characteristics and other biochemical markers on anyassociations between PAPP-A levels and cardiovascular events as observedwas performed by using stepwise Cox proportional-hazards regressionmodel. All results of the continuous variables are expressed asmedian±standard deviation. The comparison between the groups wasanalysed by a t-test (two-sided). The comparison of categoricalvariables was generated by the Pearson χ² Test. p-values of <0.05 wereregarded as statistically significant. All analyses were performed withSPSS 11.5 (SPSS Inc., Chicago).

Summary of the Results

Baseline PAPP-A plasma levels indicated an average level of 14.8±13.8mIU/l (range 0.2 to 105.4). When the PAPP-A plasma levels wereassociated with common risk markers, PAPP-A concentrations did notcorrelate with TnT levels (Spearman row correlation coefficient r=0.11;P=0.16) and were similar in patients with high TnT plasma levels and inpatients with low TnT plasma levels (FIG. 14). Similarly, VEGF (r=0.08;P=0.07), and IL-10 plasma levels (r=−0.04; P=35) showed no associationwith PAPP-A plasma levels. In contrast to this, the hsCRP plasma levelswere significantly higher in patients with elevated TnT plasma levels.The bivariant correlation analysis resulted in a significant correlationbetween PAPP-A and hsCRP as well as between PAPP-A and sCD40L, althoughthe correlation coefficients were low with r=0.21 for hsCRP (P=0.001),and r=0.18 for sCD40L (P=0.001). However, when the analysis was limitedto patients without myocardial necrosis (no troponin-increase) thecorrelation between hsCRP and PAPP-A became more obvious, with an rvalue of 0.68 (P=0.001). Consequently, patients with elevated PAPP-Alevels each exhibited significantly higher hsCRP and sCD40L levels (FIG.15).

Interaction between PAPP-A Plasma Levels and Cardiac Risk

Patients were stratified in quintiles according to their measured PAPP-Alevels: (PAPP-A_(—)1)<4.5 mIU/L (n=111), (PAPP-A_(—)2) 4.5-7.5 mIU/L(n=108), (PAPP-A_(—)3) 7.6-12.6 mIU/l (n=109), (PAPP-A_(—)4) 12.7-24.0mIU/l (n=110), and (PAPP-A_(—)5)>24.0 mIU/l (n=109), respectively. Forthe initial 24-hour period, the combined endpoints mortality andnon-fatal myocardial infarction did not differ between the quintiles(P=0.69) (FIG. 16). For the 72-hours follow-up, includingperi-interventional events, the differences in the cardiac eventsbetween the levels that were reached in the quintiles reached statisticsignificance (P=0.019). During the 30-days and 6-months follow-up, theevent-rate-curves continued to diverge from one another leading tohighly significant differences between the quintiles both at 30 days(P=0.008), and 6 months follow-up (P=0.004). For the 6-months follow-updata the post hoc analysis of the PAPP-A quintiles using a logisticregression model led to the result that only the upper two PAPP-Aquintiles (4. quintile: P=0.034; 5. quintile: P=0.002) differedsignificantly from the first PAPP-A quintile serving as a reference. Inagreement with these results, the receiver-operatingcharacteristics-curve-analysis verified a threshold value of 12.6 mIU/1PAPP-A for the maximised predictive value (FIG. 17).

Stratification According to PAPP-A Status

Based on the above mentioned results the study population wasdichotomised according to the calculated threshold value of 12.6 mIU/1leading to 219 patients with elevated PAPP-A levels (40.0%). In additionto higher levels of each of hsCRP and sCD40L in patients with elevatedPAPP-A plasma levels, the baseline-characteristics in patients withelevated PAPP-A plasma levels were not significantly different frompatients with low PAPP-A plasma levels (Table 5). The odds-ratios fordeath and myocardial infarction (adjusted to differences in denbaseline-characteristics) were 1.15 (95% CI 0.36-3.67; P=1.00) at 24 h,2.96 (95% CI 1.55-5.64; P=0.002) at 72 h (FIG. 18 a), 2.84 (95% CI1.55-5.22; P=0.001) at 30 days, and 2.64 (95% CI 1.55-4.50; P 0.001) at6 months (FIG. 18 b). Six-months cumulative event rates in patients withlow PAPP-A levels were 7.9%, versus 17.4% for patients with high PAPP-Alevels. These difference in the event rates were not only caused by ahigher rate of non-fatal myocardial infarction, but also by a highermortality in patients with reduced PAPP-A plasma levels (3.2% versus1.2%; P=0.098). Correspondingly, urgent processes for revascularisationincluding percutaneous coronary intervention and coronary arterialbypass-grafting were significantly higher in patients with elevatedPAPP-A plasma levels (13.6% versus 7.9%; P=0.012). Non-urgent processesfor revascularisation during the 6 months of follow-up indicated ahigher occurrence in patients with high PAPP-A plasma levels compared topatients with lower PAPP-A plasma levels (34.4% versus 19.7%; P=0.005).

Multimarker-Considerations

The inventors simultaneously measured markers of myocardial necrosis(TnT), ischemia (VEGF), inflammation (hsCRP, PAPP-A), anti-inflammatoryactivity (IL-10), and activation of platelets (sCD40L). Remarkably, thepredictive value of PAPP-A was limited to patients with elevated hsCRPlevels (FIG. 19 a). When the hsCRP plasma level was elevated (above 10mg/l) patients with a PAPP-A plasma level of above the calculatedthreshold value of 12.6 mIU/1 indicated an elevated cardiac risk fordeath and non-fatal myocardial infarction (adjusted odds-ratio 2.61[1.25-5.62]; P=0.007) (FIG. 19 a). In contrast to this, for patientswith hsCRP values below 10 mg/L, PAPP-A did not serve as a significantpredictor for the cardiovascular risk (P=0.073). In addition, thepredictive value of PAPP-A was tightly associated with the plasma levelsof the anti-inflammatory cytokine IL-10 (FIG. 19 b). When the IL-10plasma levels were above the calculated threshold values of 3.5 ng/l,patients with elevated PAPP-A plasma levels (above 12.6 mIU/l) wereprotected from an elevated cardiac risk (adjusted odds-ratio 1.40[0.60-3.23]; P<0.001) (FIG. 19 b). However, for patients with low IL-10plasma levels, PAPP-A values above 12.6 mIU/1 identified a subgroup ofpatients that suffered from a particularly high cardiovascular risk(adjusted odds-ratio 3.52 [1.71-7.23]; P 0.001). In summary, these datashow that the predictive value of PAPP-A plasma levels is importantlymodulated by the balance between pro- and anti-inflammatorycytokine-plasma levels. Importantly, the predictive value of PAPP-A wasalso visible in patients without evidences for myocardial necrosis.TnT-negative patients (threshold value 0.1 μg/l) with elevated PAPP-Alevels were at significantly higher risk compared to TnT-negativepatients with low PAPP-A levels (adjusted odds-ratio 2.72 [1.25-5.89];P=0.009) (FIG. 20 a). In contrast to this, TnT-positive patientssuffered from an elevated cardiovascular risk being independent fromPAPP-A plasma levels. The predictive value of PAPP-A was also observedfor a reduced threshold value of 0.01 μg/L for TnT (adjusted odds-ratio3.97 [1.24-12.68]; P=0.016). In patients that were negative both for TnTand sCD40L, PAPP identified a subgroup that suffered from an elevatedcardiovascular risk during 6 months of follow-up (FIG. 20 b). In orderto further derive a potentially independent prognostic significance ofindividual biochemical markers a stepwise multivariate logisticregression analysis was performed that included the biochemical markersTnT, VEGF, hsCRP, PAPP-A, IL-10 and sCD40L as well as baselinecharacteristics that led to a significant predictive value in aunivariate model. For the endpoints death and non-fatal myocardialinfarction at 30-days and 6-months follow-up, none of the establishedrisk factors was an independent predictor after introducing thedichotomised biochemical markers TnT, hsCRP and sCD40L into the model(Table 6). Thus, the stepwise multivariate analysis was limited tobiochemical markers. TnT (P=0.008) and PAPP-A (P=0.007) remainedindependently significant predictors of the outcome of the patients,whereas hsCRP lost the significance after PAPP-A was introduced into themodel (P=0.003 without PAPP-A; P=0.16 after introduction of PAPP-A)(Table 7; step I). PAPP-A remained a significant predictor of theoutcome of the patients after the inclusion of the anti-inflammatorycytokine IL-10 (Table 7; step III; P=0.006) and after the inclusion ofsCD40L as a marker of the activation of platelets (Table 7; step III;P=0.015). After the inclusion of VEGF as a marker the myocardialischemia TnT lost its predictive power for the 6-months outcome (Table7; step IV; P=0.24 after the inclusion of VEGF versus P=0.16 before theinclusion of VEGF), whereas PAPP-A remained a significant independentpredictor (P=0.014).

The simultaneous determination of biomarkers with distinctpathophysiological profiles dramatically improves the riskstratification in patients with ACS. Since the PAPP-A, PlGF, and sCD40Llevels are relatively stable and no specific sample conditions arerequired for PAPP-A, PlGF, and sCD40L, these markers appear to besuitable for the routine clinical use. Although inherent limitations formarkers remain that are not specific for the coronary arteries and/orthe myocardium, PAPP-A, PlGF and sCD40L could represent an importanttool for the diagnostic and therapeutic stratification of patients withACS without evidence for myocardial necrosis.

TABLE 5 Base line characteristics according to the PAPP-A status PAPP-APAPP-A low high p-value n 328 219 male 70.2% 71.9% 0.62 Age 60.5 ± 1162.2 ± 10.4 0.39 troponin T ≧ 0.1 μg/l 36.4% 39.2% 0.23 VEGF > 300 ng/l50.8% 54.3% 0.43 CRP ≧ 10.0 μg/l 37.3% 56.2% <0.001 IL-10 < 3.5 ng/l57.3% 53.4% 0.38 sCD40L > 5.0 μg/l 33.4% 51.4% <0.001 ST-segmentdepression 45.1% 53.6% 0.062 T-wave inversion 51.7% 51.7% 1.00 Historyof angina > 4 weeks 55.5% 56.3% 0.64 infarction < 30 days 13.3% 12.5%0.89 infarction > 30 days 20.3% 20.6% 0.90 PTCA 17.6% 17.4% 0.75 CABG 3.4%  3.5% 0.98 Risk factors Diabetes  9.5% 10.7% 0.97 Hypertension34.6% 37.4% 0.64 Acute smoker 40.9% 42.6% 0.41 Medication beforeregistration Aspirin 98.1% 97.8% 1.00 Heparin i.v. 99.0% 98.8% 1.00Nitrates i.v. 99.5% 99.2% 1.00 Beta-blockers 63.5% 62.9% 0.94

TABLE 6 Multivariate Cox proportional-hazards-regression model forlethal and non-lethal myocardial infarction within the first 6 months offollow-up examination adjusted hazards- Variable ratio 95% CI p-valueGender 0.91 0.68 to 1.39 0.16 Age > 65 years 1.36 0.91 to 1.82 0.34Diabetes mellitus 1.22 0.83 to 1.49 0.61 Hypercholesterolemia 0.90 0.68to 1.13 0.59 Hypertension 1.00 0.89 to 1.04 1.00 History of a coronary-0.86 0.65 to 1.19 0.72 revascularisation ST-depression 1.29 0.72 to 2.310.39 troponin T > 0.1 μg/l 2.23 1.25 to 3.98 0.007 hsCRP > 10.0 mg/l2.03 1.11 to 3.59 0.018 PAPP-A > 12.6 mIU/l 2.33 1.30 to 4.17 0.005

TABLE 7 Multimarker examinations - stepwise multivariate/logisticregression model for lethal and non-lethal myocardial infarction withinthe first 6 months of follow-up examination regression- Variablecoefficient B SE wald P-value Exp(B) 95% CI step I CRP 0.38 0.28 1.820.16 1.49 0.86 to 2.59 TnT 0.72 0.27 6.88 0.008 2.07 1.21 to 3.56 PAPP-A0.83 0.27 9.01 0.007 2.13 1.24 to 3.68 step II CRP 0.20 0.29 0.46 0.501.44 0.67 to 2.17 TnT 0.75 0.27 7.44 0.006 2.13 1.24 to 3.69 PAPP-A 0.780.28 7.70 0.006 2.18 1.25 to 3.78 IL-10 −0.76 0.29 6.99 0.008 0.47 0.26to 0.82 step III CRP 0.20 0.30 0.47 0.49 1.23 0.68 to 2.19 TnT 0.66 0.285.51 0.019 1.93 1.12 to 3.35 PAPP-A 0.70 0.29 5.60 0.015 2.05 1.18 to3.32 IL-10 −0.86 0.28 9.29 0.002 0.42 0.24 to 0.74 SCD40L 0.90 0.30 9.610.002 2.45 1.39 to 4.32 step IV TnT 0.36 0.31 1.38 0.24 1.43 0.79 to2.60 PAPP-A 0.69 0.29 5.85 0.014 2.01 1.14 to 3.49 IL-10 −0.84 0.28 8.660.003 0.43 0.25 to 0.76 SCD40L 0.86 0.29 8.76 0.003 2.37 1.34 to 4.18VEGF 0.78 0.33 5.63 0.018 2.29 1.14 to 4.18

The invention claimed is:
 1. A method for diagnosing an acutecardiovascular disease comprising: (a) obtaining a biological samplefrom a patient to be analyzed; (b) measuring a concentration ofplacental growth factor (PlGF) in the sample; (c) comparing the resultsobtained in (b) for the biological sample with a PlGF concentration inat least one reference sample; (d) using the comparison to diagnose anacute cardiovascular disease.
 2. The method of claim 1, wherein theacute cardiovascular disease is chosen from unstable angina, myocardialinfarction, acute coronary syndromes, coronary arterial disease, andheart insufficiency.
 3. The method of claim 1, further comprisingadministering a therapy if an acute cardiovascular disease is diagnosed,the therapy comprising administering at least one statin or at least oneinhibitor of a glycoprotein IIb/III-receptor.
 4. The method of claim 3,wherein the at least one inhibitor of a glycoprotein IIb/III-receptor isabciximab.
 5. The method of claim 1, further comprising measuring aconcentration of at least one additional marker selected from PAPP-A,soluble CD40-ligand (sCD40L), troponin T (TnT), MPO, NT-proBNP, VEGF,BNP, and IL-10 in the sample.
 6. The method of claim 5, wherein adiagnosis of acute cardiovascular disease is indicated by: (i) aconcentration of PlGF above a reference concentration of about 27.0 ng/land a concentration of PAPP-A above a reference concentration of about12.6 mIU/l; (ii) a concentration of PlGF above a reference concentrationof about 27.0 ng/l and a concentration of sCD40L above a referenceconcentration of about 5.0 μg/I; (iii) a concentration of PlGF above areference concentration of about 27.0 ng/l and a concentration of TnTabove a reference concentration of about 0.1 μg/l; (iv) a concentrationof PlGF above a reference concentration of about 27.0 ng/l and aconcentration of VEGF above a reference concentration of about 300 ng/l;or (v) a concentration of PlGF above a reference concentration of about27.0 ng/1 and a concentration of IL-10 below a reference concentrationof about 3.5 ng/l.
 7. A method of determining a prognosis for an acutecardiovascular disease comprising: (a) obtaining a biological samplefrom a patient to be analyzed; (b) measuring a concentration of PlGF inthe sample; (c) comparing the results obtained in (b) for the biologicalsample with a PlGF concentration in at least one reference sample; (d)using the comparison to determine the prognosis of an acutecardiovascular disease.
 8. The method of claim 7, wherein the acutecardiovascular disease is chosen from unstable angina, myocardialinfarction, acute coronary syndromes, coronary arterial disease, andheart insufficiency.
 9. The method of claim 7, further comprisingadministering a therapy for an acute cardiovascular disease if anegative prognosis is determined, the therapy comprising administeringat least one statin or at least one inhibitor of a glycoproteinIIb/III-receptor.
 10. The method of claim 9, wherein the at least oneinhibitor of a glycoprotein IIb/III-receptor is abciximab.
 11. Themethod of claim 7, further comprising measuring a concentration of atleast one additional marker selected from PAPP-A, soluble CD40-ligand(sCD40L), troponin T (TnT), MPO, NT-proBNP, VEGF, BNP, and IL-10 in thesample.
 12. The method of claim 11, wherein a negative prognosis for anacute cardiovascular disease is indicated by: (i) a concentration ofPlGF above a reference concentration of about 27.0 ng/l and aconcentration of PAPP-A above a reference concentration of about 12.6mIU/l; (ii) a concentration of PlGF above a reference concentration ofabout 27.0 ng/l and a concentration of sCD40L above a referenceconcentration of about 5.0 μg/l; (iii) a concentration of PlGF above areference concentration of about 27.0 ng/l and a concentration of TnTabove a reference concentration of about 0.1 μg/l; (iv) a concentrationof PlGF above a reference concentration of about 27.0 ng/l and aconcentration of VEGF above a reference concentration of about 300 ng/l;or (v) a concentration of PlGF above a reference concentration of about27.0 ng/l and a concentration of IL-10 below a reference concentrationof about 3.5 ng/l.
 13. A method of monitoring therapy for an acutecardiovascular disease in a patient, comprising: (a) obtaining abiological sample from a patient to be analyzed; (b) measuring aconcentration of PlGF in the sample; (c) comparing the results obtainedin (b) for the biological sample with a PlGF concentration in at leastone reference sample; (d) using the comparison to monitor therapy for anacute cardiovascular disease to detect an improvement or lack thereof ina pathophysiological condition; as indicated by a change in theconcentration of the PlGF; and (e) adjusting the therapy for an acutecardiovascular disease, if necessary, in light of the improvement orlack thereof detected in (d).
 14. The method of claim 13, wherein theacute cardiovascular disease is chosen from unstable angina, myocardialinfarction, acute coronary syndromes, coronary arterial disease, andheart insufficiency.
 15. The method of claim 13, wherein the therapycomprises administering at least one statin or at least one inhibitor ofa glycoprotein IIb/III-receptor.
 16. The method of claim 15, wherein theat least one inhibitor of a glycoprotein IIb/III-receptor is abciximab.17. The method of claim 13, further comprising measuring a concentrationof at least one additional marker selected from PAPP-A, solubleCD40-ligand (sCD40L), troponin T (TnT), MPO, NT-proBNP, VEGF, BNP, andIL-10 in the sample.
 18. The method of claim 17, wherein a lack ofimprovement in a pathophysiological condition is indicated by: (i) aconcentration of PlGF above a reference concentration of about 27.0 ng/land a concentration of PAPP-A above a reference concentration of about12.6 mIU/l; (ii) a concentration of PlGF above a reference concentrationof about 27.0 ng/l and a concentration of sCD40L above a referenceconcentration of about 5.0 μg/l; (iii) a concentration of PlGF above areference concentration of about 27.0 ng/l and a concentration of TnTabove a reference concentration of about 0.1 μg/l; (iv) a concentrationof PlGF above a reference concentration of about 27.0 ng/I and aconcentration of VEGF above a reference concentration of about 300 ng/l;or (v) a concentration of PlGF above a reference concentration of about27.0 ng/l and a concentration of IL-10 below a reference concentrationof about 3.5 ng/l.
 19. A method of treating an acute cardiovasculardisease, comprising: (a) obtaining a biological sample to be analyzed;(b) measuring a concentration of PlGF or soluble CD40-ligand (sCD40L) inthe sample (c) comparing the results obtained in (b) for the biologicalsample with a PlGF concentration or a sCD40L concentration in at leastone reference sample; and (d) administering at least one inhibitor of aglycoprotein IIb/III-receptor if the concentration of PlGF measured in(b) is greater than the PlGF concentration in the reference sample in(c), or if the concentration of sCD40L measured in (b) is greater thanthe sCD40L concentration in the reference sample in (c).
 20. The methodof claim 19, wherein the at least one inhibitor of a glycoproteinIIb/III-receptor is abciximab.
 21. The method of claim 19, wherein theat least one inhibitor of a glycoprotein Milli-receptor is administeredif the concentration of PlGF in the biological sample is above areference concentration of about 27.0 ng/l of if the concentration ofsCD40L in the biological sample is above a reference concentration ofabout 5.0 μg/l.
 22. The method of claim 19, wherein the acutecardiovascular disease is chosen from unstable angina, myocardialinfarction, acute coronary syndromes, coronary arterial disease, andheart insufficiency.